2011 Rustbelt RNA Meeting
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Poster abstracts

1. Title not available online - please see the printed booklet.

Amarpreet K. Ahluwalia (Department of Chemistry, The Pennsylvania State University), Jennifer L. Wilcox, Philip C. Bevilacqua (Department of Chemistry, The Pennsylvania State University)

Abstract not available online - please check the printed booklet.

2. Model systems for the investigation of CELF1 involvement in early heart development

Yotam Blech-Hermoni (Cell Biology Cleveland Clinic and Department of Molecular Biology and Microbiology CWRU), Andrea N Ladd (Cell Biology Cleveland Clinic)

Abstract:
The vertebrate heart develops from a simple, straight, “primary heart tube” to a heart consisting of ventricles, atria, septa, and valves. An important aspect of developmental gene regulation is alternative splicing. In vertebrates, where an increasing number of genes are multi-exonic, alternative splicing can give rise to multiple mature mRNA species from a single precursor mRNA. CELF1 (CUG-BP1- and ETR3-Like Family member 1) is a regulator of alternative splicing. During embryonic development, CELF1 transcript is detected in the myocardial cell layer of the developing heart, while the protein is expressed predominantly in the myocardial nuclei. Protein expression peaks between days 3 and 8, after which protein levels remain low into the postnatal period. These data suggest a temporally and spatially important role for CELF1 in cardiomyocytes during a period in which cardiomyocytes undergo significant waves of proliferation and migration, as part of cardiac morphogenesis.
The objective of this work was to identify ex vivo systems that will allow us to investigate the involvement of CELF1 in early heart development. We can isolate primary cardiomyocytes from the developing heart, knock down CELF1, and investigate molecular interactions identified through biochemical approaches. We are also currently investigating a whole heart culture system, with the aim of observing the progress of developmental events following siRNA knockdown of CELF1 in tissue ex vivo. Current work is focused on optimizing transfection conditions for knockdown and overexpression of CELF1 in the cultured hearts. In addition, biochemical approaches to identify targets of CELF1 splicing regulation activity are being carried out in parallel. These targets will then be investigated using the ex vivo systems described here.

Keywords: CELF1, alternative splicing, heart development

3. Title not available online - please see the printed booklet.

Peter C. Breen (Department of Chemistry, The Pennsylvania State University, PA 16802), Barbara L. Golden (Department of Biochemistry, Purdue University, West Lafayette, IN 47907)

Abstract not available online - please check the printed booklet.

4. Cell-type Specific Alternative Polyadenylation in Arabidopsis Roots

Jingyi Cao (Botany Department, Miami University)

Abstract:
Alternative PolyAdenylation (APA) is defined as the use of more than one polyadenylation site in a gene thus producing different transcripts with potentially different coding sequences and/or regulatory cis-elements. Dynamic APA has been discovered in different tissues, developmental stages and environmental responses in regulating gene expression across animals and plants under certain conditions, such as cell programming, cancer cell development in mammalian cells and Arabidopsis flowering time control. However, the influences of APA during cell differentiation and organogenesis in plants are not understood.

Due to its simple organization, Arabidopsis root becomes a tractable system to study plant organogenesis and cell differentiation since the cell types of the root tip can be clearly defined layers. The available collection of GFP-labeled specific cell types in Arabidopsis root makes it possible to isolate each cell type by fluorescence activated cell sorting (FACS). APA profiles of the transcriptomes of differential cell types will be revealed by using a large-scale deep sequencing protocol we developed that specifically targeting the junctions of the 3’-UTR and poly(A) tails. By applying this FACS approach plus the sequencing platform, we are testing a hypothesis that APA contributes to the change in gene expression and thus plays a role in cell differentiation of Arabidopsis root. Some preliminary results of this work will be presented.

Keywords: alternative polyadenylation, fluorescence-activated cell sorting, Arabidopsis root development

5. The Ribosomal Databse Project: Tools and sequences for rRNA analysis.

Benli Chai (Ribosomal Database Project, Center for Microbial Ecology, Michigan State University), Jordan A. Fish (Ribosomal Database Project, Center for Microbial Ecology, Michigan State University), Qiong Wang (Ribosomal Database Project, Center for Microbial Ecology, Michigan State University), James M. Tiedje (Ribosomal Database Project, Center for Microbial Ecology, Michigan State University), James R. Cole (Ribosomal Database Project, Center for Microbial Ecology, Michigan State University)

Abstract:
The ribosomal RNA gene is the most important molecular marker for studying microbial phylogeny because of its universal nature and the presence of conserved sequence regions facilitating PCR primer design. The current high-throughput sequencing revolution has greatly expanded the use of rRNA in exploring and accurately cataloging microbial diversity in complex natural environments. The Ribosomal Database Project (RDP; http://rdp.cme.msu.edu) provides data and services for analyzing data derived from rRNA genes. With its analysis tool suite, private user workspace (myRDP), and monthly-updated database of aligned and annotated quality-controlled public rRNA sequences (1,921,179 as of September 2011), RDP is widely used by the microbial research community in studies of microbial ecology, evolution, environmental engineering, and in understanding the diversity of life. The RDP recently added fungal 28S rRNA gene reference sequences, along with a hand-vetted fungal taxonomy (kindly provided by C. R. Kuske, A. Porras-Alfaro and their co-workers). Fungi are important in health, food, and biogeochemical cycling. Fungal rRNA sequence can be rapidly placed into this fungal taxonomy by using the RDP Classifier, an implementation of a Naïve Baysian machine learning algorithm. The RDP Classifier is already a standard tool for high-throughput bacterial rRNA sequence taxonomic assignment. In addition, the RDP Pyrosequencing Pipeline offers an easy-to-use web-based tool suite to automate computationally intensive analyzing tasks. The Pipeline takes raw reads through initial processing steps to hierarchical clustering into OTUs or taxonomic assignment using the RDP Classifier. Several common ecological metrics are provided, including Chao1, Shannon Index and rarefaction. Results are made available in formats suitable for common statistical and ecological packages. Other RDP tools include Sequence Match for finding nearest neighbors; Library Compare for determining differentially represented taxa between two libraries; RDP Probe Match for determining taxonomic coverage of primers and probes; Tree Builder for rapid phylogenetic tree construction; and Browsers that provide entry to the public sequences.

Keywords: rRNA, pyrosequencing, phylogeny

6. Comparison of insertional RNA editing in Myxomycetes

Cai Chen (Biophysics Graduate Program, The Ohio State University, Columbus, OH, USA), David Frankhouser (Department of Physics, The Ohio State University, Columbus, OH, USA), Ralf Bundschuh ( Departments of Physics and Biochemistry, Biophysics Graduate Program, and Center for RNA Biology, The Ohio State University, Columbus, OH, USA)

Abstract not available online - please check the printed booklet.

7. Comparison of Transcription using Short Single, Double, and Snapback DNA Templates

Stephanie Clack (Chemistry, John Carroll University), Tanya Lahood (Chemistry, John Carroll University), Oksana Kozlovskaya (Biology, John Carroll University), Michael P. Martin (Biology, John Carroll University)

Abstract:
Standard in vitro transcription assays use templates that are made of linear, double-stranded DNA. In this study we used novel templates that possessed a double-stranded promoter for T7 RNA polymerase recruitment, but the remainder of each DNA construct contained three different downstream variations. The control template utilized a linear, completely double-stranded DNA template. This was compared to two templates of the same length where one template strand was self-complementary and formed a hairpin (snapback), whereas the other strand possessed no self-complementary and remained single-stranded. All three templates produced RNA that was similar in length. We compared the kinetics of transcription at different temperatures in order to determine if there is preferential transcription of the three DNA variations at each temperature.

Keywords: transcription, siRNA, kinetics

8. SC35 and SF2/ASF Regulate Stress-Responsive Alternative Splicing of MDM2

Daniel F. Comiskey, Jr. (The Ohio State University), Ravi K. Singh (The Ohio State University), Aixa S. Tapia-Santos (The Ohio State University), Dawn S. Chandler (The Ohio State University)

Abstract:
The MDM2 oncogene encodes a protein that negatively regulates p53 by targeting it for proteasome-mediated degradation. Through the induction of DNA damage and in cancer, MDM2 is alternatively spliced into a variety of isoforms. The MDM2-ALT1 isoform, comprised of exons 3 and 12, is observed in over 85% of rhabdomyosarcomas and is generated in cells in response to genotoxic stress. MDM2-ALT1 lacks a p53 binding domain and abrogates full-length MDM2 from binding p53 by sequestering it. This leads to the stabilization of p53, causing cell cycle arrest and/or apoptosis. Paradoxically, the mouse homolog Mdm2-Alt1 has been shown to accelerate tumorigenesis in a mouse model, indicating a potential role in cancer.

In order to understand the mechanisms by which MDM2 is alternatively spliced, we have developed an in vitro splicing system using MDM2 minigenes and normal and cisplatinum-treated HeLa nuclear extracts. The MDM2 3-11-12 minigene, comprising regions conserved between mouse and human MDM2, predominantly excludes exon 11 under cisplatinum treatment both in vivo and in vitro. Using ESEfinder 3.0, we identified putative binding sites for splicing regulators SC35 and SF2/ASF in exon 11 of the MDM2 minigene. We then performed a series of mutations in the predicted binding sites for SC35 and SF2/ASF. Disrupting the SC35 binding sites in exon 11 leads to greater exclusion of exon 11 under normal conditions. This is consistent with the canonical role of SC35 as a positive regulator of splicing. However, destroying the SF2/ASF binding site promotes the inclusion of exon 11 even under damaged conditions, indicating a negative role for this protein. To further understand the role of SF2/ASF, we performed immunodepletion of SF2/ASF in normal and damaged nuclear extracts using our wild-type MDM2 construct. Our results show that depleting SF2/ASF leads to the inclusion of exon 11 under damaged conditions, a pattern similar to the SF2/ASF site mutant. We are currently assaying the effects of SC35 depletion on the splicing of the minigene. Overall, our results will provide an insight into the regulation of MDM2 alternative splicing by SC35 and SF2/ASF and its importance in cancer.

Keywords: MDM2, Alternative Splicing, SC35, SF2ASF

9. Non-catalytic embryonic dADAR isoform is evolutionarily-conserved in Drosophila but not translated into protein during embryogenesis

John Cook (Zoology, Cell and Molecular and Structural Biology Program, Miami University), Lea Chhiba (Zoology, Cell and Molecular and Structural Biology Program, Miami University), Dana Doctor (Zoology, Cell and Molecular and Structural Biology Program, Miami University), Jack Vaughn (Zoology, Cell and Molecular and Structural Biology Program, Miami University)

Abstract:
Adenosine Deaminases Acting on RNA (ADARs) function to deaminate (edit) some adenosines to inosines in selected pre-mRNAs. The single-copy ADAR gene in Drosophila melanogaster (dADAR) produces two major mRNA transcript classes, termed full-length and truncated. The catalytic full-length class has been functionally well characterized as being most active in the post-pupal brain. In contrast, virtually nothing is known about the function of the non-catalytic truncated “embryonic” isoform, which in D. melanogaster has been fully sequenced and shown to contain a complete open reading frame, a stop codon in intron 6, and a poly(A)-tail. Here, we show via 3’-RACE that this mRNA transcript class is present in every Drosophila species studied, extending back to those diverging from D. melanogaster 40 million years ago. In every species studied, the sequenced transcripts terminate in intron 6, have a stop codon, and a poly(A)-tail. Utilization of Westerns employing a polyclonal anti-dADAR antibody produced using full-length protein shows that the full-length isoform class in D. melanogaster encodes a protein with the expected molecular weight at every developmental stage. Quantification of these results shows that this isoform is most abundant between 0 and 4 h of embryo development, then gradually declines during embryogenesis. Unexpectedly, the truncated isoform does not appear to encode a protein during any embryonic stage of development in D. melanogaster. This was a surprise, since this mRNA isoform class is generated in every species studied, and is highly abundant throughout embryogenesis, suggesting that it has a conserved function. These observations suggest that the truncated dADAR mRNA transcript class may function in some unknown way at the RNA level, perhaps playing a role in regulation of full-length isoform editase function, an isoform class which has virtually no catalytic activity during embryogenesis despite its demonstrated existence.

Keywords: RNA editing, dADAR mRNA isoforms, regulation of editase function

10. Characterization of FY, an orthologue of yeast 3’ end processing factor Pfs2p, in plant polyadenylation using tethering assay and 3’ RACE.

Lavanya Dampanaboina (Plant and Soil Sciences, University of Kentucky), Dr. Arthur G Hunt (Plant and Soil Sciences, University of Kentucky)

Abstract:
Polyadenylation is an essential post-transcriptional modification resulting in a mature mRNA from all RNA polII transcripts in eukaryotes. Three cis-elements - FUE (Far upstream element), NUE (near upstream element) and cleavage site (CS) - guide the process of cleavage and polyadenylation with the help of multi-subunit protein complexes CPSF (cleavage and polyadenyation specificity factor), CstF (cleavage stimulation factor) along with cleavage factors and poly(A) polymerase (Zhao, Hyman et al. 1999). FY is a WD repeat protein, a 3’ end processing factor and is one of the subunits of the plant polyadenylation machinery. It is an ortholog of yeast essential processing factor Pfs2p that is involved in assembling different polyadenylation factors in cleavage and polyadenylation process (Ohnacker, Barabino et al. 2000). Yeast Pfs2p null mutants are lethal while Arabidopsis FY null alleles are embryo lethal and deleterious to growth in Nicotiana (Henderson, Liu et al. 2005).

FY is a part of the autonomous pathway, one of the flowering pathways regulating the transition from vegetative phase to the reproductive phase in Arabidopsis. FY targets FLC for repression by associating with another nuclear RNA binding protein, FCA. The FY-FCA complex also auto-regulates FCA expression by the selection of proximal polyadenylation site resulting in a truncated FCA protein (Henderson, Liu et al. 2005). Recent reports also show that FY regulates the polyadenylation site choice of FLC (Feng, Jacob et al. 2011). Here we employed a tethering assay to test the functional role of FY in 3’ end processing. To accomplish this MS2 coat protein is fused with FY and MS2 binding sites are added to the 3’UTR of the GFP reporter. To visualize the functional assay, transient studies were done using Agroinfiltrations in N.benthamiana. Further 3’RACE is used to confirm the visual results from transient studies. Results in these lines will be presented.

References:
1. Feng, W., Y. Jacob, et al. (2011).

Keywords: 3end processing, Tethering assay, RACE

11. The tRNA intron endonuclease from the haloarchaeal organism, Haloferax volcanii, plays a broad role in RNA metabolism.

Richard N. Nist (Department of Microbiology, The Ohio State University), Anice Sabag-Daigle (Department of Microbiology, The Ohio State University), Charles J. Daniels (Department of Microbiology, The Ohio State University)

Abstract:
The tRNA intron endonuclease from the haloarchaeal organism, Haloferax volcanii, recognizes its substrate using a structure-specific element, the bulge-helix-bulge motif (BHB). In this motif the cleavage sites are located in two three-nucleotide bulge loops that are separated by four base pairs. This enzyme does not require the presence of mature tRNA structures, a property that distinguishes it from its eukaryal counterparts. The lack of a requirement for mature tRNA structure predicts that the archaeal enzyme could act on molecules other than intron-containing tRNAs and have a broader role in RNA metabolism in this organism. Sequence analysis of the H. volcanii genome revealed three intron-containing tRNAs: tRNATrp, tRNAMet and tRNAGln; each possesses the characteristic BHB motif. We also noted that BHB motifs were present in the helices formed by the interactions of the spacer regions flanking the 16S and 23S rRNA in the predicted rRNA primary transcript. These regions are analogous to the RNase III sites in bacterial rRNA primary transcripts and suggested that the tRNA intron endonuclease may function in the maturation of the rRNA primary transcript in H. volcanii. To address this question we have examined the consequence of over-expressing a catalytically inactive form of the H. volcanii tRNA intron endonuclease protein, EndA*, in vivo using a newly developed plasmid expression system. Expression of the EndA* mutant protein leads to an accumulation of intron-containing pretRNAs indicating the mutant has a dominant effect and that these cells are deficient in endonuclease activity. Northern analysis probing RNAs from the rRNA operon indicate that several intermediates are affected by EndA* expression; one notable example is the presence of 16S-tRNAAla intermediates resulting from decreased cleavage at predicted sites flanking the 16S rRNA. Unexpectedly, we also observed that a small RNA resulting from a cleavage-ligation reaction of sequences flanking the 23S rRNA was absent when EndA* was expressed. This suggests that the tRNA intron splicing system (endonuclease + ligase) is responsible for the formation of a novel spliced RNA from the rRNA primary transcript.

Keywords: Intron processing, tRNA metabolism, Archaea

12. Gene expression analyses of MHC-CELFΔ mice implicate CELF-mediated alternative splicing in contractile gene regulation

Twishasri Dasgupta (Department of Cell Biology, Lerner Research Institute, Cleveland Clinic), Andrea N. Ladd (Department of Cell Biology, Lerner Research Institute, Cleveland Clinic)

Abstract:
Members of the CUGBP and ETR3 like factor (CELF) protein family regulate alternative splicing in the developing heart. The requirement for CELF splicing activity in heart muscle has been demonstrated with MHC-CELFΔ transgenic mice. In this model, CELF activity is inhibited in the heart muscle postnatally via the expression of a nuclear dominant negative protein (NLS-CELFΔ) under an α-MHC-promoter. MHC-CELFΔ mice develop cardiomyopathy characterized by alternative splicing defects, enlarged hearts and severe contractile dysfunction. A higher expressing “severe” line develops a severe form of cardiomyopathy, while a lower expressing “mild” line exhibits a milder form of the phenotype. Interestingly, both cardiac function and size recovers with age in the mild line without the loss of NLS-CELFΔ or change in the endogenous CELF protein expression. The severe line does not recover, perhaps because of extensive, irreversible muscle loss within the first few weeks.
In this study, expression profiles of wild type and MHC-CELFΔ hearts at 3 weeks were compared using gene expression microarrays. Gene ontology and pathway analyses identified calcium signaling and contraction as the most affected processes. Differences in expression levels were validated for several contractile and calcium regulating genes by real time RT-PCR and Western blot. To determine whether gene expression changes recover in the mild line with function as the animals age, we compared wild type and transgenic mice at 24 weeks. Even though cardiac function returns to normal in the mild line, surprisingly few transcripts return to wild type levels of expression and most remain affected. This suggests an additional compensatory pathway is activated. Additional array experiments are being performed with 24 week-old mice to compare the global expression patterns in wild type versus transgenic to elucidate these compensatory pathways.

Keywords: CELF proteins, Alternative splicing, Transgenic mice

13. Characterizing ribosome biogenesis and assembly using mass spectrometry

Romel P. Dator ( Department of Chemistry, University of Cincinnati), Patrick A. Limbach ( Department of Chemistry, University of Cincinnati)

Abstract not available online - please check the printed booklet.

14. Segmental isotopic protein labeling approach to study Protein-RNA interactions via NMR

Aaron Decker (University of Cincinnati, Department of Chemistry), Sheng Liu (University of Cincinnati, Department of Chemistry), Mary Anne Refaei (Ohio State University, Departments of Chemistry and Biochemistry), Pearl Tsang (University of Cincinnati, Department of Chemistry)

Abstract:
In solution, the interaction of RNA with a large protein such as human lysyl aminoacyl tRNA synthetase (hLysRS) is difficult to study by NMR due to inherent problems with significant NMR resonance overlap. Using the enzyme Sortase, derived from the gram-positive bacteria S. Aureus, a single domain of a multi-domain protein can be selectively and isotopically labeled using controlled ligation techniques. We have employed sortase for this via a previously established method1,2. Using this approach, hLysRS RNA binding can be investigated on a domain-by-domain basis. Since the structure and RNA binding properties of the N-terminal domain of hLysRS remain poorly characterized3, we are specifically interested in labeling the first domain of hLysRS for NMR study. The ability to generate a segmentally labeled form of this enzyme that allows selective observation of the N-terminal domain while part of the larger enzyme represents an important step towards understanding the RNA-binding properties of this domain. Towards this end, we have worked towards generation of a two-domain construct of hLysRS consisting of its first two domains, the N-terminal and anticodon binding (ACB) domains; these are also the most essential for overall RNA binding. For optimal NMR studies of the structure and RNA binding properties of the first domain, we have generated a two-domain version of hLysRS consisting of an 15N-labeled N-terminal domain enzymatically ligated to the unlabeled ACB domain. The RNA binding and preliminary structural analysis of the actual two –domain, segmentally labeled protein generated via sortase ligation will be presented and discussed.

References:
1. Hongyuan Mao, Scott A. Hart, Amy Schink and Brian A Pollok. (2004) Journal of Analytical Chemistry, 2670-2671.

2.  Mary Anne Refaei, Al Combs, Douglas J Kojetin, John Cavanagh, Carol Caperelli, Mark Rance, Jennifer Sapitro, Pearl Tsang. (2011) Journal of Biomolecular NMR, 49: 3-7.

3. Min Guo, Micheal Ignatov, Karin Musier-Forsyth, Paul Schimmel, Xiang-Lei Yang. (2008) PNAS Vol 105, No. 7 2331-2336.

Keywords: Sortase, Protein Engineering, NMR-RNA titrations

15. A kinetic approach to the mechanism for nucleic acid renaturation

Alice A. Deckert (Allegheny College), Brittany Rauzan, Rachel Cave, Lesley Sevcik, Kara Ostrofsky, Betsy Whitman (Allegheny College)

Abstract:
The activation parameters, &Delta*H and &Delta*S, for renaturation of two octamers (5'-CACAGCAC/GTGCTGTG-3' and 5'-CACGGCTC/GAGCCGTG-3') of DNA, the homomorphous RNA, and complementary hybrids were measured as a function of the sodium chloride concentration (0.01 to 1.0 M). The change in enthalpy of activation for the RNA renaturation reactions increased from approximately zero kcal/mol to 10-15 kcal/mol (depending on sequence) with increasing salt concentration whereas the activation parameters for the DNA renaturation reactions initially decreased from 5-10 kcal/mol to nearly zero kcal/mol before increasing slightly to 3-5 kcal/mol. The change in entropy of activation mirrored these trends. The &Delta*S for the RNA renaturation reactions increased from -30 to -40 cal/mol K at 0.01 M [NaCl] to +10 to +20 cal/mol K at 1.0 M [NaCl]. In contrast, the value of &Delta*S was negative (approximately -30 cal/mol K at the minimum) or nearly zero at all salt concentrations for the DNA renaturation reactions. The trends in activation parameters for the hybrids were intermediate to these two cases. The data are interpreted in terms of a reaction path that includes disruption of intrastrand stacking and hydration interactions, association of the two complementary strands, and rehydration of the transition state.

Keywords: kinetics, renaturation, mechanism

16. Dissecting the role of Has1 in 60S ribosome biogenesis

Jill A Dembowski (Biological Sciences, Carnegie Mellon University), Benjamin Kuo (Biological Sciences, Carnegie Mellon University), John L Woolford (Biological Sciences, Carnegie Mellon University)

Abstract:
Ribosomes are ubiquitous nanomachines that translate mRNA into proteins. Ribosome biogenesis occurs in a precisely controlled step-wise manner during which preribosomal RNAs (pre-rRNAs) undergo highly regulated structural changes, endonucleolytic cleavages and exonucleolytic processing coupled to hierarchical assembly of ribosomal proteins to generate the mature 60S and 40S ribosomal subunits. DEAD-box RNA helicases are a highly conserved group of energy consuming enzymes that utilize ATP to unwind short RNA duplexes and to remodel ribonucleoproteins. In this study, I am taking a comprehensive look at the function of the essential DEAD-box RNA helicase, Has1 in 60S ribosome biogenesis. Has1 copurifies with 66S preribosomes and is required for two consecutive pre-rRNA processing steps to generate the mature 60S ribosomal subunit. To more carefully dissect the function of Has1 in these steps, I am working to precisely map when Has1 enters preribosomes, what factors it depends on to enter preribosomes, what factors and ribosomal proteins depend on Has1 to stably associate with preribosomes or to leave preribosomes, and when Has1 exits preribosomes. We have also identified protein partners of Has1, which may function as Has1 cofactors, activators, or repressors or that may be protein targets that are either stabilized or released from preribosomes by Has1. Additionally, to understand the exact role of Has1 in pre-rRNA processing, cross-linking and analysis of cDNAs is being used to determine the specific RNA binding sites of Has1 on the preribosome. In the future, I plan to use RNA structure probing to understand how Has1 alters preribosomal architecture to generate the mature 60S subunit. Taken together, this systematic approach will allow us to understand the role of this assembly factor in 66S processing steps and will reveal new insights into the mechanisms of 60S ribosome biogenesis.

Keywords: helicase, preribosome, pre-rRNA processing

17. Modulation of Splicing Events to Achieve an Intermediate Mouse Model

Catherine E. Dominguez (Molecular, Cellular, and Developmental Biology Graduate Program, The Ohio State University), Thomas W. Bebee (Molecular, Cellular, and Developmental Biology Graduate Program, The Ohio State University), Dawn S. Chandler (Pediatrics, Nationwide Childrens Research Institution)

Abstract not available online - please check the printed booklet.

18. Title not available online - please see the printed booklet.

Min Dong (Botany Department, Miami University), Guoli Ji (Automation Department, Xiamen University, China), Q. Quinn Li (Botany Department, Miami University), Chun Liang (Botany Department, Miami University)

Abstract not available online - please check the printed booklet.

19. A sensitive assay for quantifying full-length and endonucleolytic decay products of nonsense-containing β-globin mRNA

Julie A Dougherty (Molecular and Cellular Biochemistry, Ohio State University), Roshan Mascarenhas (Molecular and Cellular Biochemistry, Ohio State University), Daniel R Schoenberg (Molecular and Cellular Biochemistry, Ohio State University)

Abstract:
The appearance in erythroid cells of stable 5’-truncated products of nonsense-containing β-globin mRNA (1) was the first evidence for endonuclease cleavage in NMD. These decay products are independent of PTC position and their appearance is linked to the PMR1 endonuclease(3). They were originally detected using S1 protection; however, this assay is time-consuming, insensitive, difficult to quantify and difficult to apply to large numbers of samples. To monitor changes in full-length and decay products following overexpression and knockdown of proteins in the decay process we developed a highly sensitive assay that combines 5’-linker ligation and 5’ RACE termed MBRACE (3). The first steps involve cap hydrolysis with TAP or Dcp2, and selection of poly(A) RNA. The 5’-monophosphate ends are then ligated to an RNA linker and the product is next converted into an oligo(dT)-primed cDNA. Finally, the modified MBRACE qPCR is performed using a forward primer matching the ligated RNA oligo, a reverse primer downstream within β-globin mRNA and two dual-labeled molecular probes that are specific for the junction between the linker sequence and either the uncapped 5’-end of the full-length mRNA or the 5’ end of one of the identified decay intermediates. Data are normalized to β-actin as determined by SYBR Green qPCR. Data will be presented showing the applicability of this to studying the impact of altering the location of the PTC on the degree of endonuclease cleavage and of knocking down Upf1 and SMG6 on this endonuclease-mediated process.

References:
1. Lim S-K et al. (1992) Molecular and Cellular Biology. 12:3; 1149-1161.
2. Bremer et al. (2003) RNA. 9: 1157-1167.
3. Lasham A et al. (2009) Nucleic Acids Research. 38:3 e19

Keywords: nonsense-mediated mRNA decay, endonucleolytic decay

20. 3′ UTR context dependent differential expression of miRNA targeted genes

Jeysabith Ebron (Department of Biological, Geological and Environmental Sciences, Cleveland State University), Jagjit Singh (Department of Biological, Geological and Environmental Sciences, Cleveland State University), Kavleen Sikand (Department of Biological, Geological and Environmental Sciences, Cleveland State University), Andrew Sedowski (Department of Biological, Geological and Environmental Sciences, Cleveland State University), Jinani E Slaibi (Department of Biological, Geological and Environmental Sciences, Cleveland State University), Girish C. Shukla (Department of Biological, Geological and Environmental Sciences, Cleveland State University)

Abstract:
Noncoding microRNAs (miRNA) constitute less than 1% of all human genes and have been implicated in regulatory control of a large number of human genes. Although a lot is known about the miRNA biogenesis and function, a clear understanding of the control of translational repression mediated by miRNAs by targeting 3′ untranslated region (UTR) of mRNAs has remained elusive. A single miRNA has potential to target multiple genes or bind to multiple sites within one target mRNA. Similarly, many miRNAs have potential to target one mRNA. However, how miRNA and target mRNA hybrids and their surrounding sequences and structures play a role in translational repression and/or target mRNA stability is not clear. This study is aimed at understanding the control of miR 488* targeted gene expression by using a 3′ UTR target library constructed in luciferase reporter. Our data show that the miRNA exerts different levels of repression in different 3′ UTR context, and bioinformatic studies confirm that the numbers of binding sites are proportional to the level of repression.

Keywords: miRNA, 3-UTR

21. Zinc-dependent regulation of a natural antisense transcript

Kate M. Ehrensberger (Molecular Genetics, The Ohio State University), Amanda J. Bird (Molecular Genetics, The Ohio State University)

Abstract not available online - please check the printed booklet.

22. Developing an in silico pipeline for sequence analysis of small RNAs in human pathogens

Will Foran (Biology)

Abstract:
Antisense transcription of mRNA regions is known to be capable of affecting gene expression. To assess if there is evidence for this in Plasmodium falciparum, mixed blood stage sRNA was sequenced and mapped to the genome. Contigs meeting length and expression metrics that could also be mapped complementary to gene regions were ranked. This was then correlated to stage specific gene expression annotated in PlasmoDB.

Regions upstream of highly expressed contigs, including both regions mapped antisense to intergenic and intragenic genomic positions, were also examined for common motifs using a discriminative algorithm. The motif elucidated within 1000nts upstream of these regions is very similar to that found upstream of tRNAs.

Keywords: Plasmodium falciparum, sRNA

23. How expression of antibiotic resistance genes is triggered in bacteria: a structural study of the ykkCD tetracycline-responsive riboswitch RNA

Alysa M. Frank (Ball State University), Krystal Roark (Ball State University), Timea Gerczei (Ball State University)

Abstract:
Riboswitches are RNA aptamers that bind to small molecules thus causing allosteric structural changes and regulating gene expression. Most identified riboswitches specifically recognize the metabolic product of the gene to be regulated. Once metabolite concentrations reach a threshold it will bind to the riboswitch causing a structural change. In most cases this binding will turn off transcription or translation of the metabolite-producing gene. The ykkCD riboswitch appears to recognize the antibiotic tetracycline and up-regulates expression of an efflux pump that exports toxic drugs from the bacterial cell. The heart of riboswitch function is allosteric structural change triggered by metabolite binding. In this work we present nucleic acid footprinting studies in order to (1) map where tetracycline is binding to the RNA; (2) compare these findings with mutagensis studies performed by pervious members of the lab. Once these data have been collected we then want to map the structural change that take place upon binding antibiotic tetracycline to determine whether gene expression is regulated at the transcription or translation stage. Taken together these data provide insight into how expression of antibiotic resistant genes are triggered - a key step in fighting bacterial pathogens.

Keywords: regulation, footprinting, gene expression

24. Title not available online - please see the printed booklet.

Lihua Fu (Department of Chemistry, University of Cincinnati), Patrick A. Limbach (Department of Chemistry, University of Cincinnati)

Abstract not available online - please check the printed booklet.

25. Title not available online - please see the printed booklet.

Bobby Gaffney (Plant and Soil Sciences, University of Kentucky), Arthur Hunt (Plant and Soil Sciences, University of Kentucky), Randy Dinkins (USDA-FAPRU), Patrick Thomas (Plant and Soil Sciences, University of Kentucky)

Abstract not available online - please check the printed booklet.

26. Title not available online - please see the printed booklet.

Kirk W. Gaston (Department of Chemistry, University of Cincinnati), Gabriela Phillips (Department of Microbiology, University of Florida), Valerie de Crecy-Lagard (Department of Microbiology, University of Florida), Patrick A. Limbach (Department of Chemistry, University of Cincinnati)

Abstract not available online - please check the printed booklet.

27. A novel role for full-length dADAR in 5'-UTR intron splicing regulation of rnp-4f pre-mRNA during Drosophila embryogenesis

G. Girija Lakshmi (Zoology, Cell and Molecular and Structural Biology Program, Miami University), Sushmita Ghosh (Zoology, Cell and Molecular and Structural Biology Program, Miami University), Gabriel Jones (Zoology, Cell and Molecular and Structural Biology Program, Miami University), Bridgette Rawlins (Zoology, Cell and Molecular and Structural Biology Program, Miami University), Jack Vaughn (Zoology, Cell and Molecular and Structural Biology Program, Miami University)

Abstract:
The Drosophila rnp-4f gene encodes a splicing assembly factor that dimerizes U4- and U6-snRNPs during spliceosome formation. 5’-UTR pre-mRNA intron processing results in two major isoforms, “long” (unspliced) and “short” (alternatively spliced). The long isoform has a secondary structure in which an intron pairs with adjacent highly evolutionarily-conserved exon 2 to form a stable 177-nt stem-loop. The coding potential for the two isoforms is identical, raising interesting questions as to the control mechanism and functional significance of this 5’-UTR intron splicing decision. It is known that the unspliced isoform localizes largely in the developing fly central nervous system, as do dADAR mRNAs, and that dADAR uses dsRNAs for substrate during deamination of particular adenosines to inosines in pre-mRNAs. These observations suggested an hypothesis in which dADAR protein may bind directly to the rnp-4f pre-mRNA stem-loop and inhibit splicing. To test this hypothesis, RNA electrophoretic mobility shift assay (REMSA) was carried out using in vitro transcribed stem-loop RNA incubated with embryo protein extract. Two RNA-protein complexes are detected by shifted RNA bands, suggesting involvement of two different proteins. Protein extract from a dADAR null mutant fly line results in only one shifted band, and recombinant dADAR results in a band shift. These observations suggest that dADAR protein may in part regulate intron splicing. To determine if unspliced mRNA levels are correlated with presence of dADAR protein during embryo development, qRT-PCR was carried out using protein extracts from wild-type and the dADAR mutant. A dramatic decrease in unspliced mRNA levels occurs in the dADAR mutant, a finding consistent with the REMSA results. These observations demonstrate a novel non-catalytic role for dADAR protein in rnp-4f 5’-UTR intron splicing regulation. We are now attempting to identify the regulatory protein(s) which bind to the stem-loop using MALDI-TOF technology.

Keywords: REMSA, alternative splicing regulation, non-catalytic dADAR function

28. Solid-phase synthesis of natural and unnatural branched RNA sequences

Debasish Grahacharya (Department of Chemistry), Eduardo Paredes (Department of Chemistry), Molly Evans (Department of Chemistry), Subha R. Das (Department of Chemistry)

Abstract:
The process of splicing joins the protein coding regions of transcripts (exons) and removes the non-coding region (intron) as a lariat RNA that includes a highly conserved 2'-5'-branched RNA sequence with a branch point adenosine. The lariat branched structure is debranched by debranching enzyme after which the intronic RNA can enter critical regulatory processes such as miRNA biogenesis, retrotransposition and snoRNAs biogenesis pathways. The significance of branched RNAs in these critical regulatory processes have led to significant efforts to obtain branched RNA, yet access either to the natural conserved sequences or the ability to readily modify sequences have been elusive. We describe a straightforward strategy for the solid-phase synthesis of branched RNAs using a photolabile protecting group for reagentless and selective unmasking of an internal 2'-hydroxyl for branch synthesis. We describe the synthesis of the four photoprotected phosphoramidite compounds required for the synthesis of the conserved adenosine branched structure and three branch point analogues, guanosine, cytosine and uracil. Our method allows for rapid access to branched RNAs of any sequence and other synthetic modifications. A preliminary investigation of debranching enzyme with these branched RNAs is presented. We envision that our approach to branched RNA synthesis will aid in the molecular level understanding of the central role branched RNAs play in debranching, splicing and related critical regulatory processes.

Keywords: lariat, branched RNA, splicing

29. Analysis of the “interactome” of a long non-coding RNA

Lalith S. Gunawardane (Center for RNA Molecular Biology, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA), Bing Zhang (Center for RNA Molecular Biology, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA), Farshad Niazi (Center for RNA Molecular Biology, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA), Saba Valadkhan (Center for RNA Molecular Biology, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA)

Abstract:
Long non-coding RNAs (lncRNA) are a novel and highly understudied class of regulatory factors and their potential role in various biological aspects has unknown. To gain insight into the relationship between the function of a long non-coding RNA and the complement of cellular factors with which it interacts, we have taken a combined in vivo mutagenesis-pull down approach. We have shown that overexpression of a long non-coding RNA in mesenchymal cells leads to their differentiation into neurons. Using a battery of truncation mutants, we have been able to determine the regions in the RNA which are dispensable for its reprogramming ability versus those which are absolutely required for its function. Analysis of the complement of proteins which interact with each mutant in vivo will enable us to define the contribution of each observed interaction to the reprogramming function of the lncRNA and ultimately, define the minimal functional motifs which mediate its function.

Keywords: Long non-coding RNA

30. Thermodynamic analysis of the ATP binding pocket for a bacterial DEAD-box protein

Lauren Hammell (Chemistry Department, Allegheny College), Ivelitza Garcia (Chemistry Department, Allegheny College)

Abstract not available online - please check the printed booklet.

31. Biogenesis of Mammalian microRNAs by a Non-Canonical Processing Pathway

Mallory A. Havens (Department of Cell Biology and Anatomy, Chicago Medical School at Rosalind Franklin University), Ashley A. Reich (Biology Department, Lake forest College), Dominik M. Duelli (Department of Cellular and Molecular Pharmacology, Chicago Medical School at Rosalind Franklin University), Michelle L. Hastings (Department of Cell Biology and Anatomy, Chicago Medical School at Rosalind Franklin University)

Abstract:
Canonical microRNA biogenesis requires the Microprocessor components, Drosha and DGCR8, to generate precursor-miRNA, and Dicer to form mature miRNA. The Microprocessor is not required for processing of some miRNAs, including mirtrons, which are excised from introns by the spliceosome and subsequently cleaved by Dicer to form mature miRNAs. In this study, we examine the processing of putative human mirtrons and demonstrate that, although some are splicing-dependent, as expected, the predicted mirtrons, miR-1225 and miR-1228, are produced in the absence of splicing. Remarkably, biogenesis of these splicing-independent mirtron-like miRNAs, termed “simtrons”, requires Drosha but not the other canonical miRNA biogenesis components, DGCR8, Dicer, Exportin-5 or Argonaute 2. Both simtrons and mirtrons function in silencing of target transcripts as demonstrated by luciferase assays and interact with all of the argonaute proteins of the RISC complex as demonstrated by immunoprecipitation assays. These findings reveal a non-canonical miRNA biogenesis pathway that can produce functional regulatory RNAs.

Keywords: microRNA biogenesis, splicing, mirtron

32. Title not available online - please see the printed booklet.

Francine M. Jodelka (Department of Cell Biology and Anatomy, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA.), Anthony Hinrich (Department of Cell Biology and Anatomy, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA.), Kate McCaffrey (Department of Cell Biology and Anatomy, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA, Department of Biology, DePaul University, Chicago, IL.), Jennifer J. Lentz (Neuroscience Center, LSU Health Sciences Center, New Orleans, LA, USA), Frank Rigo, Frank Bennett (Isis Pharmaceuticals, Carlsbad, CA.), Michelle L. Hastings (Department of Cell Biology and Anatomy, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA.)

Abstract not available online - please check the printed booklet.

33. Conserved cis elements and modified trans factors dictate MDM2 alternative splicing

Aishwarya G Jacob (MCDB, OSU), Ravi K Singh

Abstract not available online - please check the printed booklet.

34. Identification of novel regulators of SMN2 exon 7 splicing in spinal muscular atrophy

Francine M. Jodelka (The Chicago Medical School, Rosalind Franklin University of Medicine and Science, Department of Cell Biology and Anatomy, North Chicago, IL; U.S.A.), Claribel D Wee (The Chicago Medical School, Rosalind Franklin University of Medicine and Science, Department of Cell Biology and Anatomy, North Chicago, IL; U.S.A.), Michelle L Hastings (The Chicago Medical School, Rosalind Franklin University of Medicine and Science, Department of Cell Biology and Anatomy, North Chicago, IL; U.S.A.)

Abstract:
       Promoting SMN2 exon 7 inclusion is a promising therapeutic strategy for SMA. Some members of the SR protein family of splicing factors have been found to control alternative splicing of exon 7. However, a comprehensive survey of all SR proteins in the context of exon 7 splicing has not been performed.

       We knocked down each member of the SR protein family (SRSF 1-12) independently in cells through transfection of siRNAs. In a reciprocal experiment, individual SR proteins were overexpressed in cells by transfection of cloned, human cDNAs into HeLa and HEK293-T cells. Changes in exon 7 inclusion in SMN2-derived transcripts were quantitated by RT-PCR and PAGE. SR proteins that were found to inhibit exon 7 inclusion were knocked down in human SMA patient cell lines and the effect on SMN protein levels was assessed by western blot.

       Knock down of individual SR proteins revealed novel regulators of SMN2 alternative splicing. Lowered expression of some proteins correlates with an increase in exon 7 inclusion while lowered expression of other members of the SR protein family correlates with a reduction in inclusion. Overexpression of these proteins confirmed that modulation of exon 7 inclusion was due to specific SR protein abundance. Knock-down of inhibitors of exon 7 inclusion resulted in an increase in SMN protein in SMA patient fibroblasts.

       SR proteins are known to influence alternative splicing, and in this family, we have identified a number of novel regulators of SMN exon 7 inclusion. SR proteins may be promising candidates for targeted inhibition; there is a high level of redundancy amongst spliceosome components; they have sequence-specific binding activities, and therefore, SR protein bioactivity can be potentially modified with high degrees of specificity and sensitivity. Consequently, manipulating SR protein activity is a viable therapeutic approach to SMA that could pose a reduced risk of treatment toxicity.

Keywords: SMN2, SMA, splicing

35. The development of methods for the site-specific labeling of spliceosomal proteins for use in single-molecule studies

Matthew L. Kahlscheuer (Department of Chemistry), Ramya Krishnan (Department of Chemistry), Nils G. Walter (Department of Chemistry)

Abstract:
Spliceosomes are multi-megadalton ribonucleoprotein (RNP) complexes responsible for catalyzing the removal of noncoding introns from eukaryotic precursor messenger RNA (pre-mRNA) transcripts and ligating the flanking coding exon sequences to produce a mature messenger RNA (mRNA). Spliceosomal assembly and catalysis require a highly dynamic coordination of proteins and RNAs with the purpose of producing mature mRNA that can be properly transcribed by the ribosome. Due to the complexity of the process and a lack of suitable tools, there is still much unknown about the assembly of the spliceosome. Single molecule fluorescence microscopy tools have recently been developed for studies of spliceosome assembly and dynamics of the pre-mRNA substrate throughout splicing.
In an effort to advance the study of splicing and delineate the exact timing and dynamics required to achieve splicing, we are currently investigating methods for the site-specific labeling of proteins, one of these methods being protein trans-splicing (PTS) using a split intein. Protein splicing is a naturally occurring process in which a protein editor, called an intein, excises itself out of a host protein in which it is embedded creating a new peptide bond between its two flanking regions, the exteins. PTS uses an artificially or naturally split intein to create a new peptide bond between flanking exteins similar to protein splicing 1. Upon incubation of the two components, protein splicing will take place, resulting in the ligation of the two extein regions. By fluorescently labeling one of the extein components, we hope to use PTS to site-specifically label several spliceosomal proteins with the ultimate goal of correlating the association and dissociation of spliceosomal components with pre-mRNA dynamics in order to gain a better understanding of the mechanism of pre-mRNA splicing.

References:
1. Vila-Porollo, M., Muir, T.W. Cell 143, 191-200 (2010)

Keywords: Splicing

36. RNA binding by the 26.9 kDa Trypanosoma brucei pentatricopeptide repeat protein: insights from deletion mutagenesis and in vitro selection.

Pakoyo F. Kamba (Department of Biochemistry and Molecular Biology, Michigan State University), Neil A. White (Department of Biochemistry and Molecular Biology, Michigan State University), David A. Dickson (Department of Biochemistry and Molecular Biology, Michigan State University), Charles G. Hoogstraten (Department of Biochemistry and Molecular Biology, Michigan State University)

Abstract:
Disease caused by Trypanosoma brucei (T. brucei), the pathogen underlying African Sleeping Sickness, is fatal to humans if untreated. Nevertheless, available drugs are either very toxic or have suboptimal efficacy, meaning novel therapies are needed. Accomplishing this requires understanding the structure and function of essential gene products of the parasite that are absent in the human host. One group of such targets is the sequence-specific RNA binding pentatricopeptide repeat (PPR) family, typified by tandem repeats of 35 amino acids. Most non-plant eukaryotes contain 2 to 6 PPR proteins. T. brucei, however, contains at least 36 [1], a number highly conserved across the trypanosomatids. Unfortunately, utilization of PPR proteins in drug design is hampered by the limited understanding of their structure and mechanism of RNA binding, owing in part to the difficulty in heterologous expression and low aqueous solubility of PPR proteins. Using the smallest T. brucei PPR protein, denoted KRIPP11 [1], we have investigated its RNA ligand by in vitro selection (SELEX). We have probed the sequence and chemical selectivity, minimum size of RNA, and PPR motif requirement for high affinity interaction by gel shift and fluorescence anisotropy assays. As a result, we have an enriched understanding of the KRIPP11-RNA interaction. In addition we have some progress in the crystallization and multidimensional nuclear magnetic resonance (NMR) spectroscopy of KRIPP1.

References:
1. Aphasizheva, I. et al., Mol Cell, 2011. 42(1): p. 106-17.

Keywords: T brucei, PPR, KRIPP11

37. Role of SR splicing factors in nuclear speckle organization and gene expression

David Song (Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign), Vidisha Tripathi (Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign), Zhen Shen (Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign), Supriya G. Prasanth (Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign), Kannanganattu V. Prasanth (Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign)

Abstract not available online - please check the printed booklet.

38. WebFR3D: A web application for finding, annotating, and analyzing new and recurrent RNA 3D motifs

Emil F. Khisamutdinov (Department of Chemistry and Center for Photochemical Science, Bowling Green State University), Anton I. Petrov (Department of Biological Sciences, Bowling Green State University), Craig L. Zirbel (Department of Mathematics and Statistics, Bowling Green State University), Neocles B. Leontis (Department of Chemistry and Center for Photochemical Science, Bowling Green State University)

Abstract:
Many non-coding RNAs, as well as mRNAs, form one or more structured domains, essential for function. The PDB contains atomic-resolution structures for a rapidly increasing number of large and complex RNAs, including RNase P, self-splicing introns, and ribosomal RNAs. Within these structures, a large number of 3D motifs occur, some recurrent and some unique. Modular 3D motifs correspond to hairpin, internal and multi-helix junction loops in secondary structures, and generally comprise characteristic non-Watson-Crick base-pairing, base-phosphate, and stacking interactions. In addition to mediating tertiary interactions, 3D motifs form binding sites for proteins, other nucleic acids, and small molecules and ions, and even catalytic sites of ribozymes. Different sequences can fold to form essentially the same 3D motif; recognizing sequence variants of the same RNA 3D motif is an important sub-goal of research aiming to predict RNA 3D structure starting from sequence, a major challenge of computational biology. We will describe WebFR3D, a web application for searching RNA 3D structures to find similar 3D motifs that is the on-line version of “Find RNA 3D” (FR3D); WebFR3D provides on-line access to the central features of FR3D, including geometric and symbolic search modes, without need for installing programs or downloading and maintaining 3D structure data locally. In geometric search mode, WebFR3D finds all motifs similar to a user-specified query structure. In symbolic search mode, WebFR3D finds all sets of nucleotides making user-specified interactions. In both modes, users can specify sequence, sequence–continuity, base pairing, base-stacking and other constraints on nucleotides and their interactions. WebFR3D can be used to locate potentially new hairpin, internal or junction loop motifs in 3D motifs or to find instances of known recurrent RNA 3D motifs in specific PDB files or across a carefully selected set of non-redundant, representative 3D structure files. The WebFR3D output page provides 3D super-position of the instances and alignments of their sequences, annotated with pairwise interactions. WebFR3D is available at http://rna.bgsu.edu/webfr3d.

Keywords: RNA motif

39. Biophysical characterization of ASF/SF2's interaction with splice site A7 in the HIV genome

Brent A. Kochert (Chemistry and Biochemistry Miami University ), Dr. Jeffrey D. Levengood (Chemistry and Biochemistry Miami University ), Dr. Blanton S. Tolbert (Chemistry and Biochemistry Miami University )

Abstract:
The HIV genome is synthesized as a 9kb polycistronic transcript that undergoes alternative splicing to produce the complete viral protein compliment. Within the HIV genome both donor and acceptor sites are coupled together to determine which of the over 40 transcripts are made and thus which viral proteins are produced. Host proteins are responsible for both the up regulation and down regulation of these sites and play a key role in the viral protein production. ASF (Alternative Splicing Factor) is one such protein that upregulates splicing in the HIV genome. ASF is a serine-arginine rich protein that upregulates splicing by recruiting U1 and U2 snRNP. The protein is roughly 33 kDa in its entirety and consists of three domains, a serine-arginine rich domain and two RNA recognition motifs (RRMs). The two RRM domains (RRM-1 and RRM-2) are responsible for recognition of the RNA and have been shown to bind exonic splicing enhancer (ESE) sequences ESE2 and ESE3 at splice site A7. The mechanism of interaction between ASF and splice site A7 is poorly understood, thus precludes a detailed understanding of how HIV regulates splicing activity at splice site A7. As a step towards gaining more insight, we conducted an isothermal titration calorimetry (ITC) study to understand how much each RRM domain contributes to binding. Also, electron paramagnetic resonance (EPR) studies allow us to probe us the dynamic and structural interactions between ASF and the RNA. Taken together, this work will advance our understanding of the HIV splicing mechanism and may pave the way to novel HIV therapeutics.

Keywords: HIV, alternative splicing

40. The DEAD-box protein Dhh1 promotes decapping by monitoring translocation

Carrie Kovalak (Center for RNA Molecular Biology, Case Western Reserve University), Thomas Sweet (Center for RNA Molecular Biology, Case Western Reserve University), Jeff Coller (Center for RNA Molecular Biology, Case Western Reserve University)

Abstract:
Regulation of the intimately related, conserved processes of mRNA translation and degradation is known to be critical for important processes such as viral infections, early embryogenesis, and control of gene expression by microRNAs. The predominant mRNA decay pathway in Saccharomyces cerevisiae begins with deadenylation followed by decapping, which then allows for 5'-to-3' exonucleolytic digestion of the mRNA. Previous models predicted that Dhh1-like DEAD-box proteins functioned following deadenylation to limit translation initiation and thus lead to ribosome-free mRNA that could be decapped and then degraded. In contrast, recently published work from our lab has established that all three steps of the 5’-to-3’ decay pathway occur while messages are bound by ribosomes. As we now know decay occurs on polyribosome-bound mRNA, we decided to re-evaluate Dhh1’s role in both decay and translational control.

To investigate Dhh1’s functional properties, we utilized the in vivo tethered function assay. We discovered that tethered Dhh1 could repress translation independent of decapping. The presence of tethered Dhh1 saturated a reporter message with ribosomes, indicative of a role for Dhh1 in inhibiting elongation, termination, or ribosome recycling. Further, we find endogenous Dhh1 is part of polyribosome complexes that contain slow-moving ribosomes. These results suggest that limiting translocation is a powerful stimulus for decapping. Consistent with this, when we artificially limited elongation rates by inserting a stretch of rare codons into a translating mRNA, the mRNA is converted into a Dhh1 substrate for decay. Our data collectively indicates that Dhh1 promotes decapping by both recognizing translational elongation aberrancies and limiting a post-initiation step in translation. These findings allude to the existence of an interface between mRNA translation and decay that is controlled by changes in elongation rates monitored by Dhh1-like proteins.

Keywords: decapping, translocation, translational control

41. Title not available online - please see the printed booklet.

Chun Kit Kwok (Chemistry, Pennsylvania State University), Yiliang Ding (Chemistry, Pennsylvania State University), Madeline Sherlock (Chemistry, Pennsylvania State University), Sarah Assmann (Biology, Pennsylvania State University), Philip Bevilacqua (Chemistry, Pennsylvania State University)

Abstract not available online - please check the printed booklet.

42. RRM recognition of HIV-1 splice site A7

Jeffrey Levengood (Department of Chemistry and Biochemistry, Miami University), Carrie Rollins (Department of Chemistry and Biochemistry, Miami University), Clay Mishler (Department of Chemistry and Biochemistry, Miami University), Nitika Dewan (Department of Chemistry and Biochemistry, Miami University), Brent Kochert (Department of Chemistry and Biochemistry, Miami University), Blanton Tolbert (Department of Chemistry and Biochemistry, Miami University)

Abstract:
Alternative splicing of the HIV-1 genome is necessary for translation of the complete viral proteome. Host proteins, such as hnRNP A1 and ASF/SF2, are used to regulate splicing at the various donor and acceptor sites along the genome. ASF/SF2 is a splicing activator while hnRNP A1 is a splicing silencer. One such site regulated by these cellular proteins is the conserved 3’ acceptor splice site A7 (ssA7). Silencing of splicing at this site is necessary in order to retain the Rev Responsive Element (RRE) in the adjacent tat/rev intron. The RRE is responsible for nuclear export of unspliced and partially spliced transcripts.
The ssA7 RNA structure contains three stem loops (SL1, SL2, and SL3). Binding sites for hnRNP A1 are located on all three stem loops while SL2 is the only region of the splice site which contains a binding site for ASF/SF2. Both proteins bind the RNA through their RNA recognition motif (RRM) domains. The RRM domains are a family of proteins which contain similar structures and conserved sequences for RNA recognition. Our research focuses on the manner in which these two proteins, with identical domains for RNA binding, are able to recognize different sequences and structures of RNA. Our research has focused primarily on hnRNP A1 as most of the binding sites at ssA7 are for this protein.
The binding determinants of hnRNP A1 for the RNA have been examined for each side. NMR experiments have identified residues on the RRM domains of hnRNP A1 that are involved in binding. Solving of the 3D structure of SL3 has revealed the orientation of the UAG bases that hnRNP A1 recognizes while binding studies have been done with mutations of these UAG residues. These studies confirm the importance of the hnRNP A1 binding sequence and structural features involved in molecular recognition.

References:
Saliou, J., Bourgeois, C., Avadi-Ben Mena, L., Ropers D, Jacquenet S, Marchand V, Stevenin J, Branlant C. Role of RNA structure and protein factors in the control of HIV-1 splicing. Front. Biosci. (2009) 14:2714-2729.
Mayeda, A,, Munroe, S., Caceres, J., and Krainer, A. Function of conserved domains of hnRNP A1 and other hnRNP A/B proteins. EMBO J. (1994) 13:5483-5495.
Marchand V, Méreau A, Jacquenet S, Thomas D, Mougin A, Gattoni R, Stévenin J, Branlant C. A Janus splicing regulatory element modulates HIV-1 tat and rev mRNA production by coordination of hnRNP A1 cooperative binding. J. Mol. Biol. (2002) 323:629-652.
Ding, J., Hayashi, M.K., Zhang, Y. Crystal Structure of the two-RRM domain of hnRNP A1 (UP1) complexed with single-stranded telomeric DNA. Gene Dev. (1999) 13:1102-1115.

Keywords: RRM, HIV , Splicing

43. Interactions of Human Superoxide Dismutase 2 with RNA

Elizabeth Lewis (Chemistry, John Carroll University), Ryan Schoonmaker (Chemistry, John Carroll University)

Abstract:
To corroborate and extend earlier findings that human superoxide dismutase 2 (SOD2) has the ability to bind to DNA, we used fluorescence spectroscopy to monitor the interaction of SOD2 with RNA. The tryptophan residues in free SOD2 fluoresce strongly when free, but are quenched upon binding RNA. The extent of quenching is proportional to the amount of RNA that is bound, thus we can derive binding affinities from these titrations. We have measured the affinity of SOD2 for poly(U) and poly(A) at different temperatures to determine if enthalpy contributes to the binding affinity. We anticipate determining whether SOD2 exhibits fluorescence quenching upon binding single- and double-stranded DNA as well as the salt dependence of SOD2 binding to RNA and DNA

Keywords: SOD2, fluorescence, thermodynamics

44. A new method for sequencing ribonucleic acids (RNAs) using isotope labeling and LC-MS/MS

Siwei Li (Department of Chemistry, University of Cincinnati), Patrick A. Limbach (Department of Chemistry, University of Cincinnati)

Abstract not available online - please check the printed booklet.

45. Identification of tRNA elements important for antitermination in the glyQS T box gene

Liang-Chun Liu (Department of Microbiology and Center for RNA Biology, The Ohio State University), Frank J. Grundy (Department of Microbiology and Center for RNA Biology, The Ohio State University), Tina M. Henkin (Department of Microbiology and Center for RNA Biology, The Ohio State University)

Abstract not available online - please check the printed booklet.

46. Lateral Root Development Associated with a Polyadenylation Factor in Arabidopsis

Man Liu (Department of Botany, Miami University), Xiaohui Wu (Department of Botany, Miami University), Arthur G. Hunt (Department of Plant and Soil Sciences, University of Kentucky), Qingshun Quinn Li (Department of Botany, Miami University)

Abstract:
Alternative polyadenylation plays an important role in gene expression regulation in eukaryotes. We previously reported that an Arabidopsis cleavage and polyadenylation specificity factor 30 (AtCPSF30) is involved in posttranscriptional processing to control the responses of plants to oxidative stresses, and the mutation of AtCPSF30, called oxt6, can affect the poly(A) site selection (Zhang et al., PLoS One, 3:e2410). We further found that this oxt6 mutant has drastically reduced lateral roots. Upon examination of the lateral root primordium development, the results showed that the reduction is mainly on stage I to III in oxt6. How AtCPSF30 regulates root development, however, is not known. Here, we have developed a deep sequencing method for the identification of polyadenylated RNA termini with poly(A) tags (PAT-seq), and applied this method to investigate alternative polyadenylation in the root development. PAT-seq identified 283 genes showed different alternative polyadenylation patterns in WT and oxt6. Among them, 25 are known genes that are involved in lateral root development. These findings provide candidate genes for studying post-transcriptional regulation in lateral root development.

Keywords: alternative polyadenylation, AtCPSF30, root development

47. Biophysical studies of RNA binding by the ACB domain of human lysyl aminoacyl tRNA synthetase

Sheng Liu (Department of Chemistry, University of Cincinnati), Christopher Jones (Departments of Chemistry and Biochemistry, The Ohio State University), Karin Musier-Forsyth (Departments of Chemistry and Biochemistry, The Ohio State University), Michael Howell (ProteinExpress), Pearl Tsang (Department of Chemistry, University of Cincinnati)

Abstract:
Human LysRS is a class IIb synthetase that relies upon its anticodon binding (ACB) domain for specific recognition and binding to cognate tRNA (Tamura et al., 1992). The focus of this research is to characterize ACB domain binding to various RNAs on a molecular level using NMR and other techniques. In order to characterize ACB interactions with these RNAs at high resolution, the NMR resonances of the ACB protein were first assigned using standard three-dimensional solution NMR methods using an 15N- and 13C-labeled form of this protein. With these resonance assignments, we have mapped the ACB protein surface that is affected by RNA binding via NMR chemical shift perturbation techniques. The RNA molecules studied this way include: a) an oligonucleotide corresponding to the anticodon stem loop of tRNALys,3 (‘ACSL’), b) (UUU)3 (‘oligoU’), c) (CCC)3 (‘oligoC’), d) CCCAGACCCUUUUAGUCAGUGGG (‘TLE23’), e) CCCAGACCCAAAAAGUCAGUGGG (‘TLE23-4A’), and f) CCCAGACCCCCCCAGUCAGUGGG (‘TLE23-4C’). Considerable overlap was observed in terms of the ACB surface affected by RNA binding for the ACSL, oligoU and TLE23 RNAs. These RNAs share a ‘UUU’ sequence motif that is a critical molecular determinant of RNA anticodon binding by the ACB. The TLE sequences, derived from a region 5’ to the primer binding site of the HIV-1 RNA genome, are studied relative to oligoU and ACSL RNAs because of its potential function as a mimic of the tRNALys,3 anticodon loop. The TLE was recently proposed to compete against tRNALys,3 for interaction with the human LysRS (Jones, et al., submitted) which is important for proper primer binding and reverse transcription during the HIV-1 life cycle. Details regarding how these RNAs interact with the ACB as well as possible implications of such an observed common binding surface will be provided and discussed.

References:
K. Tamura, H. Himeno, H. Asahara, T. Hasegawa and M. Shimizu (1992) In vitro study of E. coli tRNAArg and tRNALys identity elements. Nucleic Acids Research 20(9):2335-2339
C. Jones, J. Saadatmand, L. Kleiman and K. Musier-Forsyth (2011) submitted to PLOS Pathogens, Molecular mimicry of human tRNALys,3 by HIV-1 RNA genome facilitates viral replication

Keywords: Anticodon binding domain of LysRS, HIV-1, tRNAsupLys,3sup

48. Investigating the function of a candidate pre-rRNA processing endonuclease

Xin Liu (Department of Biochemistry and Molecular biology, Rosalind Franklin University), Binal N. Shah (Department of Biochemistry and Molecular biology, Rosalind Franklin University), Carl C. Correll (Department of Biochemistry and Molecular biology, Rosalind Franklin University)

Abstract not available online - please check the printed booklet.

49. Decapping dependant decay regulates lncRNA regulation

Lisa Lojek (Center for RNA Biology, Case Western Reserve University), Sarah Geisler (Center for RNA Biology, Case Western Reserve University), Jeff Coller (Center for RNA Biology, Case Western Reserve University)

Abstract:
There has recently been an explosion of information in the field of noncoding RNAs (ncRNAs) regarding the various types and regulatory functions these ncRNAs have. However, one largely overlooked aspect of ncRNA biology is how the ncRNAs themselves are regulated. Current knowledge suggests that ncRNAs are similar to mRNAs in that they possess 5’ caps and 3’ poly-A tails. Since these features play important roles in mRNA decay, we were interested to see if mRNAs and ncRNAs are degraded by similar pathways. Decapping dependent 5’-to-3’ turnover is a major form of decay for mRNA transcripts. This process begins with removal of the poly-A tail followed by removal of the cap, by a conserved decapping enzyme, Dcp2p, which allows for exonucleolytic decay of transcripts. Dcp2p is a central component of not only primary but also secondary decay pathways; for this reason we investigated the role of the decapping enzyme on turnover of ncRNAs. We performed RNA-sequencing in yeast, and we identified over 100 long ncRNAs (lncRNAs) sensitive to Dcp2p. We first confirmed the enzymatic decapping activity of Dcp2p was responsible for turnover of lncRNA. We then tested a variety of proteins known to be active in various decay pathways and found that none of the other known Dcp2p cofactors were also involved in lncRNA turnover, with the exception of Rat1p (the nuclear exonuclease) and Xrn1p (the cytoplasmic exonuclease). Thus, this represents a completely novel decay pathway for these lncRNA. One third of the identified lncRNAs were associated with genes that were repressed under our growth conditions. Many of these inducible genes were part of larger metabolic pathways, for example four genes within the galactose response pathway had lncRNAs associated with them. Interestingly, we saw that presence of a lncRNA negatively influenced expression of the associated gene. Specifically, induction of GAL mRNA expression upon addition of galactose to the growth media was strongly impaired when the lncRNA could not be cleared from the cell. This led us to a model where presence of a lncRNA can act to suppress a locus, and that clearance of the lncRNA by Dcp2p can abrogate suppression. Since Dcp2p is a conserved enzyme, this may represent a fine tuning mechanism for transcription of normally repressed loci for many higher eukaryotes.

Keywords: non-coding RNA, decapping

50. Multiple functions of Thg1-like proteins in Dictyostelium discoideum

Yicheng Long (Department of Biochemistry, The Ohio State University), Jane E. Jackman (Department of Biochemistry, The Ohio State University)

Abstract not available online - please check the printed booklet.

51. The role of polyadenylation in seed germination

Liuyin Ma (Depts. of Plant and Soil Sciences, University of Kentucky), Allan Bruce Downie (Depts. of Horticulture,University of Kentucky), Arthur G. Hunt (Depts. of Plant and Soil Sciences, University of Kentucky)

Abstract:
Many groups have reported that seeds contain a sizeable and diverse population of stored mRNAs, and that this pool of stored mRNAs can be the “source” of de novo protein production before the onset of transcription early in germination since 1970s(1,2,3). Most importantly, Harris and Dure(2) suggested that some proportion of the stored mRNA present in cotton seed was polyadenylated shortly after the initiation of germination. In addition, it has been reported that germination is insensitive to more general inhibitors of RNA polymerase II but sensitive to poly (A) polymerase inhibitors(4,5). All of these provide a possible suggestion that unadenylated stored RNA may have function in seed germination upon reactivating by cytoplasmic polyadenylation.

To test this hypothesis, we focused on providing evidence to prove the following hypothesis: 1) unadenylated RNA exists in seed. 2) a cytoplasmic polyadenylation machinery exists in plant. 3) unadenylated stored RNA becomes adenylated RNA during seed germination. Using microarray experiments and Illumina sequencing, we have identified mRNAs that are under- or unadenylated; these results support the first hypothesis. Using a bioinformatic approqach, a number of Arabidopsis orthologs of proteins involved in cytoplasmic polyadenylation in animals have been identified. T-DNA insertion mutants affected in several of these are currently being studied; progress in this characterization will be presented. Finally, an experimental system to study the adenylation of stored RNAs during germination is being developed.

References:
1.Ajtkhozhin, M. A., K. J. Doschanov, and A. U. Akhanov. 1976. Informosomes as a storedform of mRNA in wheat embryos. FEBS Lett 66:124-6.
2.Harris, B., and L. Dure, 3rd. 1978. Developmental regulation in cotton seed germination:polyadenylation of stored messenger RNA. Biochemistry 17:3250-6.
3. Ishibashi, N., D. Yamauchi, and T. Minamikawa. 1990. Stored mRNA in cotyledons of Vigna unguiculata seeds: nucleotide sequence of cloned cDNA for a stored mRNA and induction of its synthesis by precocious germination. Plant Mol Biol 15:59-64.
4.Tao, K. L., and A. A. Khan. 1976. Differential Effects of Actinomycin D and Cordycepin in Lettuce Seed Germination and RNA Synthesis. Plant Physiol 58:769-772.
5.Datta, K., L. Marsh, and A. Marcus. 1983. Early Growth of Wheat Embryonic Axes and the Synthesis of RNA and DNA. Plant Physiol 72:394-397.

Keywords: polyadenylation, seed, germination

52. A long non-coding RNA is upregulated in cancer and mediates resistance to apoptosis

Bing Zhang (Center for RNA Molecular Biology, Case Western Reserve University), Mahshid Malakootian (Center for RNA Molecular Biology, Case Western Reserve University), Farshad Niazi (Center for RNA Molecular Biology, Case Western Reserve University), Lalith Gunawardane (Center for RNA Molecular Biology, Case Western Reserve University), Saba Valadkhan (Center for RNA Molecular Biology, Case Western Reserve University)

Abstract not available online - please check the printed booklet.

53. Precursor-product relationship in the erythroid cell-specific decay of nonsense-containing β-globin mRNA

Roshan Mascarenhas (Center for RNA Biology, Department of Molecular & Cellular Biochemistry, The Ohio State University, Columbus, OH 43210), Julie A. Dougherty (Center for RNA Biology, Department of Molecular & Cellular Biochemistry, The Ohio State University, Columbus, OH 43210), Daniel R. Schoenberg (Center for RNA Biology, Department of Molecular & Cellular Biochemistry, The Ohio State University, Columbus, OH 43210)

Abstract:
In erythroid cells endonuclease cleavage is the rate-limiting step in the decay of nonsense-containing human β-globin mRNA. This appears to be a stochastic process as there is no evidence for any order of cleavage site detection and origin of stable decay products. The 5’-truncated intermediates generated during the decay process are polyadenylated and more stable than the parent mRNA. This complicated the approaches for studying the precursor-product relationship of these to full-length mRNA. In addition we needed a more sensitive and facile assay to monitor the decay process than the S1 nuclease protection assay that was used in our previous work. To address this we developed lines of K562 cells that are stably transfected with tetracycline-inducible forms of wild-type and PTC-containing human β-globin genes. These were used to quantify the precursor-product relationship of full-length mRNA and an endonuclease decay product using the newly-developed MBRACE assay. MBRACE is based on ligation of a primer onto the RNA 5’ end and amplification of the resulting product using primers to the different 5’ junctions and a 3’ globin specific primer. Using this we confirm the precursor-product relationship of full-length PTC-containing β-globin mRNA to the 5’-truncated decay product, and examined the role of a stabilizing element in the 3’-UTR in the prolonged half-life of the decay product.

Supported by PHS grant GM079707

Keywords: Nonsense-mediated decay, beta-globin mRNA

54. Use of 2-Aminopurine to detect positional ambiguity in group II single- nucleotide bulge loops

Elizabeth L. McMichael (Department of Chemistry, Allegheny College)

Abstract:
Six RNA hairpin sequences containing a single-nucleotide bulge loop with the fluorescent analog 2-aminopurine (2-AP) were optically melted in 1M NaCl and subject to fluorescent spectroscopy. Four of the bulge loops were of the group II variety, where the bulged nucleotide is identical to one of its nearest neighbors, leading to ambiguity in the exact position of the bulge. The structural similarity to adenine and the fluorescent nature and properties of 2-AP were used to analyze the uncertainty in position by inserting the fluorophore into the bulge loop on the 5’ and 3’ sides of the hairpin stem. Of the two remaining hairpins, one contained a group I bulge loop and the other was fully paired. Preliminary results show that the amount of fluorescence increases with temperature in group I bulge loops. Previously, structural probing of group II bulge loops within hairpin motifs (McCann et al. 2011) indicated that the bulged nucleotide is the one positioned farther from the hairpin loop. This study suggests that when 2-AP exists in group II bulge loops as the nucleotide closer to the hairpin loop, it exhibits behavior indicative of an unpaired nucleotide.

References:
Ballin, J.D.; Bharill, S.; Fioalcowitz-White, E.J.; Gryczynski, I.; Gryczynski, Z.; Wilson, G.M.; Site-Specific Variations in RNA Folding Thermodynamics Visualized by 2-Aminopuring Fluorescence. Biochemistry 2007, 46, 13948-13960.

McCann, M.D.; Lim, G.F.S.; Manni, M.L.; Estes, J.; Klapec, K.A.; Frattini, G.D.; Knarr, R.J.; Gratton, J.L.;Serra, M. J. Non-Nearest-Neighbor Dependence of the Stability for RNA Group II Single Nucleotide Bulge Loops. RNA 2007, 17, 108-119.

Keywords: 2-Aminopurine, Thermodynamics, Group II Bulge Loop

55. Characterization of hairpin elements in the HIV splice site A7 through isotopic segmental labeling

Charles M. Medert (Department of Chemistry and Biochemistry, Miami University), Jeffrey D. Levengood (Department of Chemistry and Biochemistry, Miami University)

Abstract:
The HIV splice site A7 (ssA7) dynamically associates with the competing host proteins heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) and alternative splicing factor (ASF) to regulate splicing activity in a down-regulatory and up-regulatory fashion, respectively. Hitherto, these associations have been described according to ssA7 secondary structure and specific protein binding sites within the construct. The mechanism by which ssA7 recruits these host proteins, however, is not well understood and may be a consequence of synergistic associations between hairpin elements within ssA7 that prompt a change in protein affinity. Thus elucidating three-dimensional structural characteristics using high-resolution NMR spectroscopy may enhance our appreciation for how HIV splicing mechanics operate. However, due to the inherent limitations of NMR analysis for large nucleic acid constructs, a segmental isotopic labeling approach was taken for studying the first stem loop (SL1) of ssA7. Herein, we engineered a restriction site within ssA7 for the endonuclease MSL1 that enabled linearization of the plasmid and subsequent translation of homogenous SL1 for later use in multi-dimensional NMR studies. Concurrently, a trans-acting hammerhead ribozyme was made to induce site-specific phosphodiester cleavage yielding an unlabeled SL2,SL3 construct. Three dimensional information for SL1 in context of its tertiary interactions with SL2,SL3 could then be collected after ligation of the two constructs. Hence, understanding the difference between free SL1 structure and SL1 in the context of ssA7 may provide insight into the likely tertiary interactions.

Keywords: HIV, Splicing, NMR

56. NMR and thermodynamic studies of the human oncogenic microRNA, human miR-34c

Grace Miner (Department of Chemistry and Biochemistry, Miami University), Blanton S. Tolbert (Department of Chemistry and Biochemistry, Miami University)

Abstract:

MicroRNAs (miRNA) are small non-coding RNAs that function as post-transcriptional regulators of gene expression. Initially, miRNAs are synthesized as long hairpin precursors that undergo a series of enzymatic processes prior to becoming biologically active. The heterogenous nuclear ribonucleoprotein A1 (hnRNP A1) was recently shown to stimulate the processing of a subset of primary miRNAs by forming direct interactions with their apical loops and other non-canonical sites [3]. Using a database search (mirbase.org), we compiled a list of miRNAs that contain the hnRNP A1 high affinity UAG binding motif located in the apical loops. Of these, we selected the human miR-34c (34c) to test the hypothesis that its apical loop is a binding site for hnRNP A1. MiR-34c has been implicated in some cancers and neurodegenerative diseases; thus, knowledge of its interaction with protein partners may have broad biomedical implications. Using electrophoretic mobility shift assays, we found that the UP1 domain of hnRNP A1 forms a stable 1:1 complex with a truncated version of human 34c. To further characterize the interaction, calorimetric titrations were performed to determine the complete thermodynamic profile at 298 K (Kd=265 nM, ΔGo= -8.92 kcal/mol, ΔHo=-39.6 kcal/mol, and -TΔSo=30.68 kcal/mol). As a step towards identifying the structural basis of UP1-34c complex stability, we used 2D NOE experiments to show that 34c folds into a stable hairpin conformation. Taken together, these data clearly show that hnRNP A1 binds to miR-34c and given the high affinity, this complex may have biological relevance.

References:
1. He, X., L. He, and G. J. Hannon.

Keywords: miRNA, hnRNP A1

57. A role for 3’-5’ polymerization in tRNA editing

Fuad Mohammad (Departments of Chemistry and Biochemistry, The Ohio State University), Maria G Abad (Departments of Chemistry and Biochemistry, The Ohio State University), Yicheng Long (Departments of Chemistry and Biochemistry, The Ohio State University), Jane E. Jackman (Departments of Chemistry and Biochemistry, The Ohio State University)

Abstract:
Transfer RNAs play a critical function in the cell as translators of information embedded in messenger RNA during protein synthesis. To ensure the fidelity of translation, each tRNA must be correctly recognized by its respective aminoacyl-tRNA synthetase and the ribosome. Therefore, tRNAs undergo various post-transcriptional modifications, some of which maintain and stabilize their universally conserved L-shaped tertiary structure. The discovery of the tRNAHis guanylyltransferase (Thg1) family of enzymes expands the repertoire of tRNA modifications to include the 3’-5’ addition of nucleotides to various tRNA substrates. Some Thg1 homologues, known as Thg1-like proteins (TLPs), catalyze the repair of 5’ truncated tRNAs, and may play a role in amending mismatches observed at the acceptor arm of several mitochondrially-encoded tRNAs in certain lower eukarya. Of the 18 mitochondrially-encoded tRNAs in Dictyostelium discoideum, 9 tRNAs contain such mismatches. Similar mismatches in other organisms, such as Acanthamoeba castellanii, have been shown to be edited to restore Watson Crick base pairing. Here, we focus on two Thg1 homologues in D. discoideum, DdiTLP3 and DdiTLP4. Using truncated tRNA substrates, both enzymes catalyze the repair of the 9 tRNAs proposed to be edited in D. discoideum, and are likely to participate in the tRNA editing mechanism. However, DdiTLP4 adds nucleotides beyond the full-length 5'-end of several tRNAs. This extra addition activity is inhibited at higher ATP concentrations, suggesting a mechanism for controlling the repair activity in vivo. Furthermore, DdiTLP4 is able to repair larger 5'-end truncations than DdiTLP3, which exhibits a more limited ability to repair these substrates and depends on the presence of a pre-activated 5'-end. Sequencing of one of the repair products reaffirms the preference for templated nucleotide addition over non-templated additions by DdiTLP3-4. These results point to an underlying 3’-5’ RNA polymerase activity of DdiTLP3-4 that has been fine tuned to participate in mitochondrial tRNA editing, and may suggest further roles for 3’-5’ nucleotide addition.

Keywords: Thg1, tRNA editing, 3-5 polymerization

58. Physical separation of capped and uncapped RNA using recombinant eIF4E-eIF4G resin

Chandrama Mukherjee (Molecular and Cellular Biochemistry, Ohio State University), Deepak Patil (Molecular and Cellular Biochemistry, Ohio State University), Brian Kennedy (Molecular and Cellular Biochemistry, Ohio State University), Daniel R Schoenberg (Molecular and Cellular Biochemistry, Ohio State University)

Abstract:
The addition of 5’ cap is the first step in pre-mRNA processing and this reaction is catalyzed by the nuclear capping enzyme associated with RNA pol II. The cap plays a central role in subsequent steps of processing steps and in translational initiation, and loss of cap was thought to irreversibly commit mRNA to decay. In contrast to this notion, our lab identified a cytoplasmic population of capping enzyme can restore the cap onto RNA with a 5’-monophosphate RNAs. Using next generation sequencing several other labs have identified populations of uncapped mRNAs in the transcriptome, as well as CAGE tags mapping to internal sites of endonuclease cleavage. Most of these studies involve ligating a primer to the free 5’-monophosphate on uncapped for subsequent physical recovery or sequencing or removing the cap prior to primer ligation for mapping to CAGE tags. By definition these are indirect approaches to studying cap dynamics, and the efficiency of this process largely depends on the ligation reaction. To facilitate work in this area we are developing a facile and quantitative method of separating capped and uncapped RNAs. Most cap-recovery based strategies used to date use a monoclonal antibody that was raised to the trimethyl cap on snRNAs or one of several bacterially-expressed forms of eIF4E none of which provide quantitative separation of capped from uncapped RNA. In order to develop a rigorous and qutiantative method to separate these two populations of RNA we examined the efficacy of several different cap binding proteins. Our results show that the most effective separation of capped from uncapped RNA is achieved by combining of eIF4E plus a fragment of eIF4G. We successfully used this technique for separation of uncapped RNA from U2OS tet-on cells stably expressing K294A, a dominant negative mutant of the cytoplasmic capping enzyme.
Supported by NIH grant GM084177

Keywords: Uncap RNA, Capping enzyme, Cap bidning protein

59. Single-Molecule Studies of HIV-1 Dimerization Initiation Sequence Kissing Interaction and its Resolution to a Stable Extended Duplex

Hansini Mundigala (Department of Chemistry,Wayne State University,Detroit. MI 48202), Jonathan Michaux (Department of Chemistry,Wayne State University,Detroit. MI 48202), Andrew Feig (Department of Chemistry,Wayne State University,Detroit. MI 48202), David Rueda (Department of Chemistry,Wayne State University,Detroit. MI 48202)

Abstract:
The Dimerization Initiation Sequence (DIS) is a conserved hairpin motif on the 5’ UTR of the HIV genome[1]. This hairpin structure plays an important role in genome dimerization by formation of a “kissing complex” between two homologous DIS sequences [2]. Understanding the kinetics of this interaction is key to exploiting DIS as a possible drug target against HIV[3]. We have developed a novel single-molecule Fluorescence Resonance Energy Transfer (smFRET) assay to study the formation kinetics of the DIS kissing complex in solution. Our data show with unprecedented clarity, the formation and dissociation dynamics of single kissing complexes as well as formation of the extended duplex conformation. We will present a complete kinetic analysis including the effect of Mg2+ and K+ ions. Our data suggests the presence of an alternative pathway for the extended duplex formation through an intermediate dimer. The observed alternative pathway and the direct pathway have similar probabilities under physiological monovalent and divalent ionic conditions. It is observed that high Mg2+ favors the newly observed alternative pathway. The formation of this intermediate dimer may be the result the H+ and Mg2+ dependent conformational dynamics of purines flanking the complementary nucleotides in DIS sequence [4,5]. The mechanistic insights gained from these experiments would represent significant progress in understanding the HIV-1 dimerization mechanism.

References:
1.Goto, T., M. Nakai, and K. Ikuta, The life-cycle of human immunodeficiency virus type 1. Micron, 1998. 29(2-3): p. 123-38.
2.Skripkin, E., et al., Identification of the primary site of the human immunodeficiency virus type 1 RNA dimerization in vitro. Proc Natl Acad Sci U S A, 1994. 91(11): p. 4945-9.
3.Berkhout, B. and J.L. van Wamel, Role of the DIS hairpin in replication of human immunodeficiency virus type 1. J Virol, 1996. 70(10): p. 6723-32.
4.Mihailescu, M.R. and J.P. Marino, A proton-coupled dynamic conformational switch in the HIV-1 dimerization initiation site kissing complex. Proc Natl Acad Sci U S A, 2004. 101(5): p. 1189-94.
5.Paillart, J.C., et al., Non-canonical interactions in a kissing loop complex: the dimerization initiation site of HIV-1 genomic RNA. J Mol Biol, 1997. 270(1): p. 36-49.

Keywords: HIV-1 DIS, smFRET, Dimerization

60. Title not available online - please see the printed booklet.

Luyen T. Nguyen (Biochemistry, Purdue University), Wai Kit Ma (Biochemistry, Purdue University), Sara C. Ess (Biochemistry, Purdue University), Elizabeth J. Tran (Biochemistry, Purdue University)

Abstract not available online - please check the printed booklet.

61. A universal initiator for co- and post-transcriptional RNA labeling and conjugation

Eduardo Paredes (Department of Chemistry, Carnegie Mellon University), Debasish Grahacharya (Department of Chemistry, Carnegie Mellon University), Subha R. Das (Department of Chemistry, Carnegie Mellon University)

Abstract:
Derivatization of RNA has greatly facilitated investigations and applications of RNA structure and function. For 5'-terminal derivatives of RNA, current methodologies involve either complex syntheses of transcriptional initiator molecules or post-transcriptional conjugations with amine or thiol reactive groups. We describe here the simple synthesis and enzymatic incorporation of 5'-propargyl adenosine into RNA. Unlike other analogs of guanosine or adenosine with a monophosphate that cause premature transcriptional arrests, 5'-propargyl adenosine as the transcriptional initiator can produce RNA that bears a 5'-alkyne. The alkyne can be used in post-transcriptional conjugations through the highly efficient Cu(I)-catalyzed azide alkyne cycloaddition reaction (click-chemistry). As click-chemistry is bioorthogonal, the conjugation reaction can be performed directly in the transcription reaction mixture. Additionally, we show that not only is 5'-propargyl adenosine a transcriptional initiator but that it can be used as an universal intermediate in near quantitative click-reactions to rapidly generate triazolyl linked adenosine analogs, each of which can then be used directly as a transcriptional initiator. These transcriptional initiators produce quantitatively 5'-functionalized RNA. This significantly expands the conditions for and simplifies access to RNA functionalized with useful molecules.

Keywords: Transcriptional priming, Conjugation, RNA modification

62. Microinjection as a tool for studying the mechanism of nuclear import of Potato spindle tuber viroid (PSTVd)

Woong June Park (Department of Molecular Biology, Dankook University, South Korea), Biao Ding (Department of Molecular Genetics, Ohio State University, Columbus, OH)

Abstract:
Nuclear accumulation of the Potato spindle tuber viroid (PSTVd) has been observed and is assumed to be essential for the replication of the viroid. However, the mechanism of nuclear import of PSTVd is still largely unknown. Many questions on the energy dependence, involvement of cytoplasmic and nucleoplasmic factors, role of the nuclear pore, structural requirements of PSTVd and many other aspects have not been addressed. To scrutinize the nuclear import of PSTVd, as a model for the viroids belonging to the family of Pospiviroidae, we tested several methods and select microinjection as a tool for study. By using this method, we traced time-dependent nuclear accumulation of PSTVd in epidermal cells of Nicotiana benthamiana. Recent data and possible application of this method will be discussed.

Keywords: PSTVd, Nuclear import, microinjection

63. Yeast tRNAHis guanylyltransferase: Kinetic investigation of nucleotide addition by a 3'-5' polymerase

Krishna Patel (Department of Chemistry& Biochemistry), Paul Yourik (Department of Chemistry& Biochemistry), Jane E.Jackman (Department of Chemistry& Biochemistry)

Abstract:
The tRNA Hisguanylyltransferase (Thg1) catalyzes an essential reaction in yeast, adding a non-templated single guanosine (G-1) to the 5’end of tRNAHis. This G-1 addition reaction is in sharp contrast to 5’-3’ addition catalyzed by other known DNA and RNA polymerases. In addition, Thg1 also catalyzes Watson-Crick (WC) base pair–dependent 3'-5' addition. Because of these unusual properties, understanding the molecular mechanism of nucleotide addition by Thg1 is of great interest. The aim of this work is to elucidate kinetic features that distinguish non-templated vs templated nucleotide addition. To do this, we exploited a kinetic framework previously developed to characterize specific steps of the non-templated G-1 addition reaction. Using single turnover conditions, we determined kinetic parameters for the nucleotidyl transfer step for all possible combinations of WC and non-WC base paired additions to various tRNAHis substrates. First, we determined that G-1 addition is the most efficient of any of the four possible N-1 addition reactions, regardless of the identity of the nucleotide at the N73 position. These data suggest that Thg1 interacts specifically with the incoming GTP nucleotide, and protein residues that participate in this recognition remain to be identified. Second, we observed a preference for addition of the correct WC base pairing nucleotide that is largely driven by an increase in the maximal rate of the reaction, rather than by inherent affinity for the incoming NTP. This mirrors the kinetic trend observed for selection of WC base pairs by canonical 5’-3’ polymerases, and suggests that the well-studied kinetic mechanism for ensuring fidelity may similarly be used by Thg1. Current studies aim to measure kinetics of nucleotide addition with TLP’S (Thg1-like proteins) from bacteria, archaea and mitochondria, which participate in tRNA 5'-end repair and are catalytically distinct from their eukaryotic counterparts.

Keywords: Thg1, tRNA, Watson-Crick base pairing

64. Characterize thermodynamics and kinetics aspects of the structural heterogeneity in the c-di-GMP riboswitch through laser assisted single molecule refolding

Bishnu P. Paudel (Department of Chemistry, Wayne State University, Detroit, MI, 48202), David Rueda (Department of Chemistry, Wayne State University, Detroit, MI, 48202)

Abstract:
RNAs play important biological roles in gene regulation and expression, RNA processing, viral replication and protein synthesis [1-3]. RNA molecules require proper secondary and tertiary structure folding to perform their biological activity [3-4]. The rugged RNA folding potential landscapes may entrap RNAs molecules in deep energy wells leading to the memory effect [5-6]. The c-di-GMP riboswitch has been shown to have population heterogeneity and the memory effect in which the interconversion between subpopulations is not observed. We have used laser assisted single molecule refolding (LASR) to introduce the interconversion between subpopulations of the c-di-GMP riboswitch and to measure the barrier heights of interconversion [6]. Our single molecule data reveals that in absence of the ligand (c-di-GMP), the riboswitch is mostly present in static undocked (sub-population1) and dynamic undocked (sub-population2) in which riboswitch is primarily in an undocked conformation with transient fluctuations to a docked structure. Molecules interconvert between sub-population1 and sub-population2 as the temperature is increased, however the percentage of interconversion even at high temperature (74˚C) is low (≤22%). These two states are separated by large energy barriers which slow interconversion. The entropic contribution is more significant than enthalpy to the interconversion barriers. When the two conformations are separated by native gel, these subpopulations redistribute to equilibrium at higher temperature. The structural elucidation of the memory effect will contribute significantly to understanding the RNA folding landscape.

References:
1.Guo, S., et al., Specific delivery of therapeutic RNAs to cancer cells via the dimerization mechanism of phi29 motor pRNA. Hum Gene Ther, 2005. 16(9): P. 1097-109.
2.Elbashir, S.M., et al., Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature, 2001. 411(6836):p. 494-8.
3.Pyle, A.M., Metal ions in the structure and function of RNA. J Biol Inorg Chem, 2002. 7(7-8): p. 679-90.
4.Woodson, S.A., Metal ions and RNA folding: a highly charged topic with a dynamic future. Curr Opin Chem Biol, 2005. 9(2): p. 104-9.
5.Zhuang, X. et al., Correlating structural dynamics and function in single ribozyme molecules. Science, 2002. 296(5572): p. 1473-6.
6.Zhao, R., et al., Laser-assisted single-molecule refolding (LASR). Biophys J, 2010.99(6): p. 1925-31.

Keywords: c-di-GMP riboswitch, memory effect, smFRET

65. Splicing Interference (SPLICEi) By Small Nuclear RNAs in Breast Cancer

Chaucola K. Pleasant (BGES, Cleveland State University), Girish C. Shukla (BGES, Cleveland State University)

Abstract:
In nuclear pre-mRNA splicing, introns are removed through the work of small nuclear RNAs (snRNAs). In the human genome there are two types of introns. Major class introns, the U2-dependent type, which are spliced by U1, U2, U4, U5 and U6 snRNAs, and the minor class introns, the U12 dependent type, which are spliced by U11, U12, U4atac, U5, and U6atac snRNAs. It has been observed that over expression of minor class spliceosomal snRNAs can have an inhibitory effect on the splicing of major class introns. To further test the concept we targeted the Human Epidermal Receptor 2(HER-2)/Neu proto-oncogene, which is over- expressed in 20-30% of breast tumors. We constructed a series of mutant human U6atac and U11 snRNAs to target introns 1, 2, 6, 8, 12 and 13 of HER-2/Neu to block nuclear pre-mRNA splicing and down regulate protein synthesis. We transfected mutant snRNAs in breast cancer cell line MDA-MB-453 to determine the efficacy of our approach on HER-2/Neu pre-mRNA splicing interference. Our preliminary data indicates variable effect on the HER-2/Neu pre-mRNA splicing and protein synthesis in cultured cells. Further experiments are in progress to confirm the efficacy of mutant snRNAs in blockading of HER-2/Neu expression.

Keywords: snRNA, splicing, Breast Cancer

66. Pri-miRNAs: the imperfect targets of the “microprocessor”

Kaycee A. Quarles (Chemistry Department at Pennsylvania State University, University Park), Christopher Wostenberg (Chemistry Department at Pennsylvania State University, University Park), Ellen Forsyth (Chemistry Department at Pennsylvania State University, University Park), Scott A. Showalter (Chemistry Department at Pennsylvania State University, University Park)

Abstract:
Found in multicellular organisms and encoded by viruses (1,2), microRNAs (miRNAs) are ~22-nucleotide-long single-stranded non-coding RNAs that participate in various developmental and differentiation processes via post-transcriptional regulation of gene expression (3-5). Despite high interest in these RNAs, the molecular mechanism of miRNA maturation by the “Microprocessor” complex—consisting of the double-stranded RNA binding protein DGCR8 in complex with the RNase III enzyme Drosha—is poorly understood. Mechanistic proposals emphasize a role of primary-miRNA (pri-miRNA) structural heterogeneity in Microprocessor binding based on the variety of bulges and internal loops present in pri-miRNA stemloops. However, atomistic structural biology has not shed insight into these mechanistic proposals due to the lack of determined pri-miRNA structures.
To begin evaluating the structural features of pri-miRNAs in solution, structure mapping using SHAPE chemistry (6) was performed on a panel of miRNAs. SHAPE reactivities indicate that many of the small imperfections predicted by mFold do not deform the helix significantly enough for the SHAPE reagent to access, suggesting that the processing proteins may not be able to recognize these imperfections either. On the other hand, electrophoretic mobility shift assays performed on a model pri-miRNA containing native imperfections binding DGCR8 showed tighter binding in comparison to binding a perfect duplex of similar length. The results suggest that DGCR8 may bind the pri-miRNA tighter than the perfect duplex due to the presence of the flanking single-strands and terminal loop; however, the interactions of DGCR8 with these pri-miRNA features are poorly defined. Defining the thermodynamics of DGCR8 binding pri-miRNA and the extent to which DGCR8 can tolerate helix imperfections will provide a framework for how the Microprocessor selects miRNA targets for processing and positions the Drosha cut site within them.

References:
1. Liang, D.; Gao, Y.; Lin, X.; He, Z.; Zhao, Q.; Deng, Q.; Lan, K., Cell Res 2011, 21 (5), 793-806.
2. Lin, H. R.; Ganem, D., Proc Natl Acad Sci U S A 2011, 108 (13), 5148-53.
3. Zeng, Y.; Cullen, B. R., Rna-a Publication of the Rna Society 2003, 9, 112-123.
4. Pfeffer, S.; Zavolan, M.; Grasser, F. A.; Chien, M. C.; Russo, J. J.; Ju, J. Y.; John, B.; Enright, A. J.; Marks, D.; Sander, C.; Tuschl, T., Science 2004, 304 (5671), 734-736.
5. Dostie, J. E.; Mourelatos, Z.; Yang, M.; Sharma, A.; Dreyfuss, G., Rna-a Publication of the Rna Society 2003, 9 (2), 180-186.
6. Wilkinson, K. A.; Merino, E. J.; Weeks, K. M., Nat Protoc 2006, 1 (3), 1610-6.

Keywords: miRNA, Microprocessor, structure

67. In-silico exploration of alternatively spliced introns in Chlamydomonas reinhardtii

Praveen Kumar Raj Kumar (Botany Department, Miami University), Chun Liang (Botany Department, Miami University)

Abstract:
Pre-mRNA splicing is one of the fundamental post-transcriptional processes in eukaryotic gene expression and regulation. Alternative Splicing (AS) occurs when different splice sites of pre-mRNA are processed to generate distinct transcript isoforms, leading to diverse proteins with functional and/or structural differences from the same genes. Pre-mRNA splicing is guided by the cis-regulatory sequence motifs buried in them; currently in mammals these are well characterized as the consensus splice sites, branch point signal and poly-pyrimidine tract. Focusing on Chlamydomonas reinhardtii, a green alga that shares both animal (e.g., flagella) and plant traits (e.g., chloroplast), we evaluate AS events and their associated cis-regulatory signals. Based on all available Sanger-based ESTs (338,243), JGI 454 cDNAs (6,317,641) and genoscope 454 cDNAs (689,548), we evaluated AS events using GMAP and PASA, and updated AUGUSTUS gene annotation by resultant PASA AS models. Among 17,163 AUGUSTUS protein-coding genes, ~49% are subjected to AS while ~17% of PASA genes shows AS evidence. As observed in other plants analyzed so far, we found the dominant AS mode is intron retention: over 51% of the AS genes retain their introns in their mature mRNAs. In comparison with the constitutively spliced introns, we found that the retained introns tend to have weaker splice sites, with less abundant G triplet (intronic splicing enhancer) and C triplet. Our data also suggests that the majority of AS isoforms are presumably functional and not Non-sense Mediated Decay (NMD) candidates. We also observed that most retained introns are less than 250 nt, which have a potency of being spliced by intron definition splicing mechanism. It suggests that short introns that are spliced by intron definition are more likely to be retained if they possess weak signals.

Keywords: alternative splicing, intron retention, chlamydomonas

68. A thermodynamic study of HIV splicing factors

Prashant V. Rajan (Department of Chemistry and Biochemistry, Miami University), Blanton S. Tolbert, PhD (Department of Chemistry and Biochemistry, Miami University)

Abstract:
The alternative splicing pathway within the HIV-1 virus plays a key role in HIV replication and viral infectivity; however splicing mechanisms in HIV are poorly understood at the molecular level. In alternative splicing, the protein-coding exons of the viral pre-mRNA are isolated from the non-protein coding introns and then ligated in various permutations, thereby resulting in the full protein complement for the virus. Genome splicing in HIV is dynamically modulated by interactions between cis and trans regulatory elements; the trans factors belong to the host’s SR and hnRNP factors. The host factor hnRNP A1 negatively regulates ssA2, ssA3, and ssA7 by binding cis silencer elements that are embedded within regions containing higher-order RNA structure. Recently, we have solved the NMR structure of ESS3, which is located at ssA7. This structure revealed unique stereochemical and tertiary properties at the A1 binding site. Based on these results, we hypothesize that structural environment modulates hnRNP A1-ESS3 interactions, which in turn may affect splicing activity. As an initial step to test that concept, this study is focused on comparing binding affinities of hnRNP A1 to different ESS3 elements across different clades of HIV. Insights from this study will allow us to better understand the splicing mechanisms that underlie HIV replication.

Keywords: Splicing, HIV, RNA

69. Title not available online - please see the printed booklet.

Bhalchandra S. Rao (Chemistry and Biochemistry, The Ohio State University), Jane E. Jackman (Chemistry and Biochemistry, The Ohio State University)

Abstract not available online - please check the printed booklet.

70. A Structural and Thermodynamic Study of Alternative Splicing of the HIV Genome

Carrie Rollins (Department of Chemistry and Biochemistry, Miami University), Jeffrey Levengood (Department of Chemistry and Biochemistry, Miami University), Clay Mishler (Department of Chemistry and Biochemistry, Miami University), Nitika Dewan (Department of Chemistry and Biochemistry, Miami University), Grace Miner (Department of Chemistry and Biochemistry, Miami University)

Abstract:
Despite advances in identifying splicing regulatory sequences in the HIV genome, the mechanism of alternative splicing has not been well defined on the molecular level. Splicing plays a critical role in the lifecycle of the virus, transforming one strand of pre-mRNA into over forty different mRNA constructs. Various combinations of acceptor and donor splice sites function to accomplish such variability, but how each splice site is utilized in competition with additional splice sites is not well understood. Host proteins such as heterogeneous ribonucleoprotein A1 (hnRNP A1) are known to downregulate splicing by binding to highly conserved acceptor splice sites A2, A3, and A7 and blocking spliceosome assembly. The protein-RNA interaction displays different levels of affinity with each acceptor site, suggesting that the structural environment may modulate complex stability. High-resolution structural information provides the foundation to define a splicing mechanism. The focus of this study is to elucidate the structures of SLISS and SLESS3 of splice site A7 as well as of SLESSV of splice site A2 using multidimensional NMR experiments. In addition we will define the mechanism of interaction these RNAs will make with the UP1 domain of hnRNP A1. Electromobility shift assays and isothermal titration calorimetry provide additional insight of the interaction to further understand this structure/function relationship.

Keywords: Alternative Splicing, HIV

71. Utilizing PNAs to target telomeric RNA regions of Plasmodium falciparum

Samantha Sanford (Department of Chemistry, Center for Nucleic Acids Science and Technology CNAST, Carnegie Mellon University), Danith Ly (Department of Chemistry, Center for Nucleic Acids Science and Technology CNAST, Carnegie Mellon University), Bruce Armitage (Department of Chemistry, Center for Nucleic Acids Science and Technology CNAST, Carnegie Mellon University), Kausik Chakrabarti (Department of Chemistry, Center for Nucleic Acids Science and Technology CNAST, Carnegie Mellon University)

Abstract not available online - please check the printed booklet.

72. WITHDRAWN

Juan E. Santiago-Torres (Integrated Biomedical Science Graduate Program, The Ohio State University), Lien Lai (Biochemistry, The Ohio State University), Venkat Gopalan (Biochemistry, The Ohio State University), Daniel R. Schoenberg (Molecular and Cellular Biochemistry, The Ohio State University)

Abstract:
WITHDRAWN

Keywords:

73. PeanutDB: integrated bioinformatics web portal for Arachis hypogaea transcriptomics

Emily Schmidt (Botany Department, Miami University), Xiaohong Duan (Institute of Plant Protection, Chinese Academy of Agricultural Sciences, China), Jie Zhang (Institute of Plant Protection, Chinese Academy of Agricultural Sciences, China), Chun Liang (Botany Department, Miami University)

Abstract:
The peanut (Arachis hypogaea), an annual herbaceous plant in the legume, is an important crop for oil production and food sources. As in July 15, 2011, there were a total of 198,156 nucleotide sequences for Arachis hypogaea in NCBI GenBank, including 39,854 Nucleotide (Core Nucleotide Sequences), 150,177 EST (Expressed Sequence Tag), and 8,125 GSS (Genome Survey Sequence). Based on previously available 73,407 Sanger ESTs, NCBI UniGene Build#1 was developed on Dec 2009, which contains 11,909 UniGene clusters. With the next generation sequencing like 454 pyrosequencing and Illumina SBS, more and more genomics resources are being generated for peanut. For example, there are a total of 596.5 million bases (3.6 GB) 454 ESTs available in NCBI SRA. Recently, Illumina paired-end sequencing of peanut transcriptome has been conducted in Institute of Plant Protection, Chinese Academy of Agricultural Sciences and generated a total of 20 GB sequence data (~ 80 million reads) for peanut. As the first public genomic database dedicated to Arachis hypogaea, PeanutDB currently focuses on the transcriptomics analysis of Arachis hypogaea. Using all aformentioned cDNA data, we have created our first release of peanut transriptome assembly consisting of 32,619 contigs. Not only we provided GO, KEGG, EC and InterproScan annotation to these contigs, but also we determined the relationship among these contigs based on sequence similarity and the potential linkage from the paired-end/clone-end read information. Obviously, our database will be an useful bioinformatics resource that will facilitate peanut genome sequencing and gene annotation in the future.

Keywords: peanut , transcriptome , database

74. How does Anti-TRAP regulate TRAP in the B.subtilis trp operon?

Shraddha Sharma (State University of New York at Buffalo), Paul Gollnick (State University of New York at Buffalo)

Abstract not available online - please check the printed booklet.

75. Splice site mutations alters the expression of intronic miRNA

Jagjit Singh (Dept of BGES, Cleveland State University), Neha Aggarwal (Dept of BGES, Cleveland State University), Kavleen Sikand (Dept of BGES, Cleveland State University), Girish C Shukla (Dept of BGES, Cleveland State University)

Abstract:
Two main classes of nuclear pre-mRNA introns, U2- and U12- types are found in metazoan genomes. Many U2-types introns codes for regulatory noncoding miRNAs. Existing in vitro studies show that processing of primary and precursor miRNA is independent of host pre-mRNA intron processing. Due to the nature of biochemical assays, these studies failed to observe a subtle variation if any, in mature miRNA production and its resultant effect on target expression. Numerous miRNA profiling studies have shown 2 to 4 fold up or down regulation, however if this differential expression exert similar effect on target gene expression is unclear. To test, if in vivo pre-mRNA processing has any subtle effect on mature miRNA production, we used a minigene containing four exons and three introns derived from MYH6 gene. The middle intron of the minigene codes for miR-208. The 5’ splice site of the miRNA containing intron was mutated from position 3 to 7. These mutants were cotransfected with a luciferase reporter containing 3’ UTR of THRAP-1, a validated target of miR-208. Our data show drastic in vivo splicing defect in positions 4 and 5 mutants. Real-time quantitative PCR of miR-208 indicates the lower level of miRNAs from these splicing defective mutant introns. Furthermore, luciferase reporter containing 3’ UTR of THRAP-1 expression was downregulated in 4 and 5 positions splice site mutants cotransfected experiments. This data suggest that splicing defective mutation affect miRNA production and its target gene expression.

Keywords: Splicing , intronic miRNAs

76. Kinetic analysis of 3'-5' nucleotide addition catalyzed by eukaryotic tRNAHis guanylyltransferase

Brian Smith (Ohio State University Department of Biochemistry)

Abstract not available online - please check the printed booklet.

77. Characterization of the mRNP unique to substrates of the nonsense-mediated mRNA decay pathway

Jenna E. Smith (Center for RNA Molecular Biology, Case Western Reserve University), Kristian E. Baker (Center for RNA Molecular Biology, Case Western Reserve University)

Abstract:
mRNA quality control mechanisms in eukaryotes ensure fidelity of gene expression and are critical for normal cellular activity. One of the most thoroughly investigated of such mechanisms, the nonsense-mediated mRNA decay (NMD) pathway, recognizes and promotes the accelerated degradation of mRNAs containing a nonsense or premature translation termination codon (PTC), thereby guarding against the accumulation of truncated polypeptides in cells. The molecular mechanism underlying the discrimination of PTC-containing mRNAs from normal mRNAs remains to be clearly established. A prevailing model for NMD postulates that translation termination is perceived as premature if it occurs distally from the mRNA poly(A) tail and its binding protein [poly(A) binding protein, PAB1], such that PAB1 can no longer promote efficient translation termination thereby allowing the NMD machinery to be recruited to the mRNA. This model is supported by observations that 5’-proximal PTCs lead to more efficient targeting of mRNA to NMD, and that tethering PAB1 proximal to a PTC rescues the mRNA from being targeted to NMD. In contrast, however, it has been shown that in yeast, Pab1p is dispensable for NMD substrate recognition, suggesting that additional factors must be important in defining a translation termination event as premature. A critical goal for understanding how NMD substrates are recognized will be to determine the specific features of a PTC-containing mRNA and its bound proteins (i.e. the mRNP) that are important in signaling to the cellular NMD machinery that translation termination is premature. We are purifying an NMD mRNP as a means to identify and characterize such features important for targeting the mRNA to the NMD pathway. Importantly, NMD is a conserved quality control pathway in all eukaryotes, and our work in the budding yeast model Saccharomyces cerevisiae will provide insights into NMD substrate recognition in metazoa, including humans.

Keywords: NMD, mRNA decay, mRNP purification

78. RNA Function Through Compartmentalization

Christopher A. Strulson (Chemistry, Pennsylvania State University), Rosalynn C. Molden (Chemistry, Pennsylvania State University; current institution Princeton University), Christine D. Keating (Chemistry, Pennsylvania State University), Philip C. Bevilacqua (Chemistry, Pennsylvania State University)

Abstract:
Compartmentalization of RNA is essential for many cellular functions such as RNP processing and assembly, gene silencing, and transcription control, and may have played a pivotal role in the emergence of life. Variation in local RNA concentration impacts numerous biomolecular functions, from binding to catalysis. However, effects of cellular compartmentalization on RNA function, including catalysis, are largely unknown. We use RNA partitioning in an aqueous two-phase system (ATPS) to mimic intracellular compartmentalization and crowding, and test the effect on the kinetics of phosphodiester bond cleavage by a series of two-piece hammerhead ribozymes. The ATPS consists of poly(ethyleneglycol) (PEG) 8 kDa, dextran 10 kDa, and reaction buffer, in which the PEG-rich and dextran-rich phases of the ATPS serve as chemically distinct compartments. Partitioning induces a 1,000-fold enrichment of long RNA into the dextran-rich phase. Local concentration of hammerhead ribozyme is controlled by varying the volume ratio of the PEG-rich and dextran-rich phases. For ribozymes with an accessible kcat/Km region, increasing the local concentration of RNA via compartmentation into the dextran-rich phase of the ATPS enhances the observed rate of cleavage. In particular, a 20-fold enhancement in cleavage rate is observed in an ATPS consisting of a 1:100 ratio of dextran to PEG-rich phase. This study demonstrates that compartmentalization can enhance RNA function, which may be important in RNA therapeutics, in vivo RNA folding, and the evolution of functional protocells.

Keywords: Compartmentalization, Catalysis

79. Title not available online - please see the printed booklet.

Krishna C. Suddala (Biophysics, University of Michigan), Arlie Rinaldi (Chemistry, University of Michigan), Catherine Eichhorn (Chemical Biology, University of Michigan), Anthony Mustoe, Jun Feng (Chemistry, University of Michigan), Hashim Al. Hashimi, Charles L. Brooks III, Nils G. Walter (Chemistry, University of Michigan)

Abstract not available online - please check the printed booklet.

80. The role of non Watson Crick base pairs in recurrent 3D motifs in the small subunit of extremophilic ribosomes

Blake A. Sweeney (Department of Biological Sciences, Bowling Green State University, Bowling Green, OH 43403), Emil F. Khisamutdinov (Department of Chemistry and Center for Photochemical Science, Bowling Green State University, Bowling Green, OH 43403), Anton Alenko (Department of Chemistry and Center for Photochemical Science, Bowling Green State University, Bowling Green, OH 43403), Dr. Craig L. Zirbel (Department of Mathematics and Statistics, Bowling Green State University, Bowling Green, OH 43403), Dr. Neocles B. Leontis (Department of Chemistry and Center for Photochemical Science, Bowling Green State University, Bowling Green, OH 43403)

Abstract:
Temperature is one of the most important environmental variables to which living organisms adapt. At the molecular level, organisms adapt to different environments by changing the sequences of their macromolecules as well as the compositions of their membranes. At high temperature, macromolecules must resist thermal denaturation, while at low temperatures, they must retain flexibility for reversible binding and catalytic action. An essential component found in all cells is the ribosome. Leveraging atomic-resolution X-ray structures of diverse ribosomes and curated sequence alignments that include psychrophiles, mesophiles, thermophiles and hyperthermophiles, we are studying how ribosomal RNAs (rRNA) evolve to adapt to different environments. Previous work has focused on sequence changes of Watson-Crick paired bases in response to extreme heat, but little work has examined cold adaptation or the roles of non-WC base pairs. The non-WC base pairs comprise about 33% all base pairs in 16S and 23S rRNA and are essential components of RNA 3D motifs, modular structural units that correspond to hairpin, internal and junction loops in secondary structures. Non-WC pairs are also critical components of RNA tertiary and quaternary interactions that stabilize 3D architectures and inter-molecular complexes. We have assembled sequence alignments of bacterial 16S rRNAs, organized by optimal growth temperature, and used them to examine sequence variations at positions that form non-Watson-Crick basepairs. We observe systematic sequence variations at many non-WC paired sites. We examined in detail the sequence variations in the Sarcin/Ricin motif, a highly structured and widespread, recurrent 3D motif. Based on these observations, we designed RNA oligonucleotides containing this motif that correspond to sequence variants found in organisms that grow at different temperature. We measured their thermodynamic properties using UV-melting and isothermal titration calorimetry techniques to elucidate principles of thermal adaptation in recurrent 3D motifs.

Keywords: Temperature adaptation, Motifs

81. Four ribosome assembly factors are required to fold and structure ITS2 in Saccharomyces cerevisiae

Jason Talkish (Carnegie Mellon University)

Abstract not available online - please check the printed booklet.

82. Investigating the non-canonical polyadenylation machinery in D. melanogaster S2 cells

Trinh T. Tat (RNA Center - Biochemistry Dept. - Case Western Reserve University), Raul Jobava (RNA Center - Biochemistry Dept. - Case Western Reserve University), Patricia Maroney (RNA Center - Biochemistry Dept. - Case Western Reserve University), Timothy Nilsen (RNA Center - Biochemistry Dept. - Case Western Reserve University)

Abstract:
Cleavage and polyadenylaton at the 3’ end of transcripts are required post-transcriptional modifications contributing to mRNA maturation in eukaryotes. This process is critical for transcription termination, nuclear export, stability and efficient translation of mRNA. It has been known that alternative polyadenylation can alter the coding sequence of the encoded protein and have an effect on how much protein is made from a transcript. By using a sensitive assay for poly(A) tail length developed in our lab followed by sequencing, we found that the Hid and Reaper transcripts in S2 cells do not contain the normal poly(A) tail compared with several controls, such as actin or Par-6. In addition, through sequencing, we found that the sequence immediately downstream of the hexanucleotide polyadenylation signal in this mRNA is the same as the genomic DNA sequence. Therefore, the Hid and Reaper pre-mRNAs are either cleaved but not polyadenylated or deadenylated. We are currently asking what are the essential cis- and trans-acting factors that cause this phenomenon. Can these cis- or trans-acting elements be recapitulated in other constitutively expressed messages in S2 cells and in mammalian cells?

References:
1. Shi Y., Giammartino D. C. D., Taylor D., Sarkeshik A., Rice W. J., Ill J. R. Y., Frank J., Manley J. L. (2009). Molecular architecture of the human pre-mRNA 3’ processing complex. Molecular Cell. 33: 365-376.
2. Nagaike T., Logan C., Hotta I., Rozenblatt-Rosen O., Meyerson M., Manley J. (2011). Transcriptional activators enhance polyadenylation of mRNA precursors. Molecular Cell. 41: 409-418.

Keywords: polyadenylation, polyA tail length assay

83. Title not available online - please see the printed booklet.

Pallavi Thaplyal (Department of Chemistry, Pennsylvania State University,University Park, PA 16802), Barbara L. Golden (Department of Biochemistry, Purdue University, West Lafayette, IN 47907), Philip Bevilacqua (Department of Chemistry, Pennsylvania State University,University Park, PA 16802)

Abstract not available online - please check the printed booklet.

84. Son is Important for Cell Cycle Progression

Keshia Torres-Munoz (Wright State University Department of Biological Sciences), Alok Sharma (Wright State University Biomedical Sciences Program), Athanasios Bubulya (Wright State University Department of Biological Sciences), Paula A Bubulya (Wright State University Department of Biological Sciences)

Abstract not available online - please check the printed booklet.

85. Btf and TRAP150 colocalize with splicing factors and exon junction complex proteins at a gene locus in situ

Sapna Varia (Biomedical Sciences PhD Program, Wright State University), Divya Potabathula (Biological Sciences, Wright State University), Athanasios Bubulya (Biological Sciences, Wright State University), Paula A. Bubulya (Biological Sciences, Wright State University)

Abstract not available online - please check the printed booklet.

86. The initial steady state kinetic characterization of a thermophilic pseudouridine synthase (Thermotoga maritima TruB)

Govardhan reddy Veerareddygari (Department of Chemistry, University of Louisville), Eugene G. Mueller (Department of Chemistry, University of Louisville)

Abstract:
Pseudouridine synthases (Ψ synthases) catalyze the isomerization of uridine (U) in RNA to pseudouridine (Ψ). Ψ synthases are classified into six different families based on sequence alignments and have a universally conserved aspartic acid residue that is essential for activity. RNA containing 5-fluorouridine ([F5U]RNA) has been used as a mechanistic probe. E. coli TruB (EcTruB) is not inhibited upon incubation with [F5U]RNA but instead handles it as a substrate. Thermotoga maritima TruB (TmTruB) on the other hand, is irreversibly inhibited by [F5U]RNA and forms an apparently covalent adduct with it. Despite this difference in behavior, cocrystals of [F5U]RNA and both EcTruB and TmTruB show a noncovalent complex in which the F5U is not only hydrated but rearranged to the C-glycoside isomer. This hydrated product was thought to result from the hydrolysis of an ester linkage between the active site Asp and C6 of the pyrimidine ring of F5U in agreement with one of the two proposed mechanisms for Ψ synthases. Later, the [18O] labeling studies on TruA, TruB and RluA, conducted by the Mueller group proved that the oxygen atom in the hydrated products of F5U comes from the solvent, rather than from the active site Asp. The interesting difference in behavior of a thermophilic versus mesophilic TruB towards [F5U]RNA demands the characterization of TmTruB and extension of mechanistic studies that were performed on its mesophilic counterpart. The initial steady state kinetic characterization of TmTruB using a new HPLC based assay is complete, and the groundwork has been laid for the mechanistic studies of TmTruB with [F5U]RNA.

Keywords: pseudouridine, fluorouridine

87. Single molecule studies of Prp24-dependent folding dynamics of the U2/U6 complex

Chandani Warnasooriya (Department of Chemistry, Wayne state University, Detroit, MI 48202), Zhuojun Guo (Department of Chemistry, Wayne state University, Detroit, MI 48202), David Rueda (Department of Chemistry, Wayne state University, Detroit, MI 48202)

Abstract:
Splicing is catalyzed by the spliceosome, which consists of five small nuclear ribonucleoprotein particles (snRNPs): U1, U2, U4, U5 and U6 and associates with several protein factors that facilitate structural rearrangements in the spliceosome [1, 2]. Proper assembly of spliceosomal components is critical for function, and thus, defects in assembly can be lethal [3, 4]. In our lab, we use Fluorescence Resonance Energy Transfer (FRET) along with single molecule detection to study the structural dynamics of the snRNA complexes and the effect of protein factors on those complexes. Our previous studies have shown that the U2/U6 complex undergoes conformational changes and has at least three conformations with three distinct FRET values [5]. Here we study the effect of Prp24, an snRNP protein, on the structural dynamics of U2/U6 complex. Our single molecule data reveals that binding of Prp24 affects the conformational dynamics of U2/U6 complex and it stabilizes the low FRET conformation. With these results we propose that Prp24 plays a major role in spliceosomal assembly and function; more specifically binding of Prp24 toggles U6 between active and inactive conformations such that it is only catalytically active when all components at activated spliceosome are present and are ready for splicing.

References:
1.Wahl, M.C., C.L. Will, and R. Luhrmann, The spliceosome: design principles of a dynamic RNP machine. Cell, 2009. 136(4): p. 701-18.
2.Will, C.L. and R. Luhrmann, Spliceosome structure and function. Cold Spring Harb Perspect Biol, 2011. 3(7).
3.Cooper, T.A., L. Wan, and G. Dreyfuss, RNA and disease. Cell, 2009. 136(4): p. 777-93.
4.Wang, G.S. and T.A. Cooper, Splicing in disease: disruption of the splicing code and the decoding machinery. Nat Rev Genet, 2007. 8(10): p. 749-61.
5.Guo, Z., K.S. Karunatilaka, and D. Rueda, Single-molecule analysis of protein-free U2-U6 snRNAs. Nat Struct Mol Biol, 2009. 16(11): p. 1154-9.

Keywords: Prp24, spliceosome, single molecule

88. Targeted LC-MS/MS Identification of tRNA Through the Detection of Unique Transitions

Collin Wetzel (Chemistry, University of Cincinnati), Patrick Limbach (Chemistry, University of Cincinnati)

Abstract:
The global identification of individual transfer ribonucleic acids (tRNAs) can prove to be very difficult, requiring rigorous sample preparation prior to analysis due to complex tertiary structures made up of several highly conserved structural motifs. Our lab has previously shown LC-MS approaches to characterize endonuclease digestion products of tRNAs through the detection of “Signature Digestion Products” (SDPs) is an ideal way for global analysis. While this is a simple and novel approach it has limitations. The use of an MS/MS approach adds a degree of selectivity to the detection aspect increasing identification confidence, decreasing sample analysis time and increases the total number of tRNA detectable through the use of one enzymatic digestion. For example, in E.coli when using RNase T1, only 28 of the 47 tRNA isoacceptors are able to be identified by LC-MS analysis of SDPs alone; MS/MS analysis increases this number to 39. Furthermore, we have discovered only 18 precursor m/z values need to be targeted to obtain enough unique transitions to detect these 39 tRNA isoacceptors. This minimal detection requirement combined with steep LC gradients and fast linear velocities yield fast sample analysis time only 9 minutes, while minimizing sensitivity losses due to MS/MS vs. MS analysis. The extremely high linear velocity used in these experiments was made possible by the utilization of solid core RP-LC technology found in the Poroshell® column provided by Agilent.

References:
Hossain, M. & Limbach, P.A. RNA 13, 295 (2007).
Hossain, M. & Limbach, P.A. Anal. Bioanal. Chem (2008).
Castleberry, M.C. & Limbach, P.A. Nucleic Acids Res. (2010)

Keywords: Oligonucleotide, tRNA, MSMS

89. Single molecule studies of the c-di-GMP riboswitch

Sharla Wood (Chemistry Department, Wayne State University), Adrian Ferre-dAmare (National Heart, Lung, and Blood Institute, NIH), David Rueda (Chemistry Department, Wayne State University)

Abstract:
Cyclic diguanylate (c-di-GMP) is a bacterial second messenger important for physiologic adaptation and virulence. The class-I c-di-GMP riboswitch is phylogenetically widespread and is thought to mediate pleiotropic genetic responses to the second messenger. Previous crystallographic, solution X-ray scattering and biochemical studies suggest that the aptamer domain of the RNA switches from an extended free state to a compact c-di-GMP-bound conformation in which two helical stacks dock side-by-side. Single molecule fluorescence resonance energy transfer (smFRET) experiments now reveal that the free RNA exists in four distinct populations that differ in their characteristic dwell times in the extended and docked conformations. In the presence of c-di-GMP and Mg2+, a population that is stably docked for >30 minutes becomes predominant. smFRET analysis of riboswitch mutants demonstrates that tertiary interactions distal to the c-di-GMP binding site strongly modulate the population structure of the RNA, even in the absence of the second messenger. These allosteric interactions accelerate ligand recognition by preorganizing the RNA, favoring rapid c-di-GMP binding.

Keywords: riboswitch, single molecule FRET, c-di-GMP

90. dsRBDs: the workhorse of the miRNA maturation pathway

Christopher Wostenberg (Chemistry Department at Pennsylvania State University, University Park), Kaycee A. Quarles (Chemistry Department at Pennsylvania State University, University Park), Scott A. Showalter (Chemistry Department at Pennsylvania State University, University Park)

Abstract:
miRNAs are small non-coding ssRNAs, ~ 22 nucleotides, which are involved in gene-regulation by base-pairing with mRNA. At least 60% of human genes are regulated by one or more miRNAs, which leads to control of cellular homeostasis (1, 2), while misregulation leads to disease states (3). Maturation occurs in two independent and spatially separated steps. First, in the nucleus, the single-stranded tail of primary miRNA is cleaved by the “Microprocessor”, composed of Drosha, an RNase III enzyme, and its cofactor DGCR8, a dsRNA binding protein (dsRBP) (4). Next, the precursor miRNA is exported to the cytosol and the terminal loop is cleaved by the RNase III enzyme Dicer aided by the dsRBPs TRBP or PACT (4). The most common protein motif in the pathway is the dsRNA binding domain (dsRBD), which prefers to bind A-form dsRNA (4, 5). A total of ten dsRBDs are involved in miRNA maturation, one in Drosha, two in DGCR8, one in Dicer, three in TRBP and three in PACT.
NMR spin relaxation and MD simulations on two of the dsRBDs in the Microprocessor, Drosha-dsRBD and DGCR8-dsRBD1, were preformed to provide further information on the molecular mechanism of binding (6). The study was motivated by EMSAs, which showed that Drosha-dsRBD does not bind dsRNA but isolated DGCR8-dsRBD1 does (6). Our results show that while loop 2 in both dsRBDs is highly dynamic, it is the increased flexibility of loop 1 in Drosha-dsRBD that potentially explains the lack of dsRNA binding in by the dsRBD. We also investigated the interactions of the two tandem dsRBDs of DGCR8 with MD simulations (7). The crystal structure along with preliminary NMR data revealed that the two dsRBDs of DGCR8 are packed against a well-defined secondary structure formed from the linker and the C-terminal tail (8), which contrasts with the flexible linker seen in PKR (9). Recent work has focused on understanding the dsRBDs in the second step of miRNA maturation, especially Dicer-dsRBD due to its high binding affinity.

References:
1. Friedman, R. C. et al., Genome Res. 2009, 19 (1), 92-105.
2. Stanczyk, J. et al., Arthrit. Rheum. 2008, 58 (4), 1001-1009.
3. Wang, Y. et al., Genet. 2008, 74 (4), 307-315.
4. Jinek, M. and Doudna, J. A., Nature 2009, 457 (7228), 405-412.
5. Tian, B. et al., Nat. Rev. Mol. Cell Biol. 2004, 5 (12), 1013-1023.
6. Wostenberg, C.et al., Biochem. 2010, 49, 10728-10736.
7. Wostenberg, C. et al., Biophys. J. 2010, 99, 248-256.
8. Sohn, S. Y. et al., Nat. Struct. Mol. Biol. 2007, 14 (9), 847-853.
9. Nanduri, S. et al., EMBO J. 1998, 17 (18), 5458-5465.

Keywords: miRNA, dsRBD, Microprocessor

91. Polyadenylation Factor PCFS4 Physically Interacts With the Loci of a Subset of Genes Involved in Arabidopsis Development

Denghui Xing (Botany, Miami Univeristy), Quinn Li (Botany, Miami University)

Abstract:
Arabidopsis polyadenylation factor, PCFS4, an ortholog of human and yeast PCF11, functions in Arabidopsis flowering time control, leaf development and phyllotaxy determination. The role of PCFS4 in flowering time control is partially mediated by FLC and correlated with the regulation of alternative polyadenylation (APA) of FCA. PCFS4 forms an in vivo complex with FY, another polyadenylation factor and a regulator of the APA of FCA, suggesting its involvement with a polyadenylation machinery that acts on the APA of FCA. The roles of PCFS4 in leaf development and phyllotaxy, however, could not be explained by the APA of FCA. We thus speculated that there must be additional genes, other than FCA, being targeted by PCFS4. To identify these targets, we compared the transcriptomes of pcfs4-1 mutant and wild type using tiling microarray assays. 68 Differentially Processed Genes (putative direct targets) in the mutant were identified and largely confirmed by RT-qPCR. To further explore if PCFS4 directly targets on the loci of those genes, we performed a ChIP assay for one of the targets, ATIM, using PCFS4-TAP recombinant protein. We found PCFS4-TAP was indeed enriched on the 3’ end of ATIM locus. A further Illumina-Seq assay following the ChIP precipitation revealed about 800 PCFS4-TAP enrichment sites on the genome. Some of the enrichment sites are indeed overlapped with the target genes of PCFS4. Analysis of the sequence features of those PCFS4-TAP enrichment sites is on going.

Keywords: Polyadenylation, Plant Development

92. A large non coding RNA is involved in the cellular stress response

BING ZHANG (RNA Center, CWRU), Lalith Gunawardane (RNA Center, CWRU), Farshad Niazi (RNA Center, CWRU), Saba Valadkhan (RNA Center, CWRU)

Abstract:
BORG is a ~2700 nucleotide long nuclear transcript that is spliced and polyadenylated but has no protein-coding capacity. It is induced when cells are exposed to stressful conditions in several different cell types, including cells of mesodermal and ectodermal origin. We use microarray to analyze the expression of factors that are involved in cellular stress response and apoptosis when we overexpressed or knocked down the lncRNA. Our analysis shows that several factors that are involved in the apoptosis pathways have been significantly changed in these two cell types. After exposure to stress, the knockdown cells exhibited an
impaired cellular stress response and a much higher death rate compared to controls, while the overexpression cells showed an elevated level of stress response and a cellular stress-resistant phenotype. Together, these data indicate that the lncRNA plays an important role in regulation of the stress response and cellular survival under stressful conditions.

Keywords: lncRNA, cellular stress

93. Comparative analysis of polyadenylation signal variations during evolution in eukaryotes

Zhixin Zhao (Botany Department, Miami University), Xiaohui Wu (Botany Department, Miami University), Q. Quinn Li (Botany Department, Miami University), Chun Liang (Botany Department, Miami University)

Abstract:
Messenger RNA Polyadenylation [Poly(A)] plays crucial molecular functions including preventing mature mRNA from degradation and recognition by the transport apparatus for cytoplasmic exportation and by the translational initiation complex for translation. In this study, we analyzed the genomes of eleven eukaryotic species (two diatoms, two green algae, red alga, ciliate, moss, spikemoss, Arabidopsis, yeast and human) for cis-elements around poly(A) sites, particularly in the Near Upstream Element (NUE) region located right before the poly(A) sites. For all species analyzed, there is a complex transition of nucleotide composition around the poly(A) sites and within the NUE region. Considering both the frequency and significance of poly(A) signals in the NUE region, while yeast and ciliate do not have much conserved signals, a triplet (UAA) and two tetramers (UAAA and GUAA) were found dominant in diatoms and red alga respectively. The green algae and humans possess high frequency and extremely conserved UGUAA and AAUAAA signals respectively. The remaining embryophytes, including moss, spikemoss and Arabidopsis, use the conserved AAUAAA signal but in low frequency (~8%), which implied that somewhat different mechanisms is used in polyadenylation process in embryophytes. The single nucleotide variants from AAUAAA suggested that the first two bases (i.e., NNUAAA) are degenerated and hence highly flexible whereas UAAA are the core part of the motif, which is consistent with wet-lab study results of polyadenylation signal efficiency. Comparing the poly(A) signals in the examined eleven species, our results suggest that AAUAAA and its variants are derived from UAA with an intermediate status of UAAA. However, the UGUAA signal might follow an independent evolutionary path.

Keywords: polyadenylation, evolution, poly(A) signal