2007 Rustbelt RNA Meeting
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Poster abstracts

1. Insights into the plant CstF complex

Amrita Bandyopadhyay (Plant and Soil Sciences, University of Kentucky), Arthur G. Hunt (Plant and Soil Sciences, University of Kentucky)

Abstract:
3’ end modification by polyadenylation is a ubiquitous feature of almost all of the eukaryotic mRNA species. This process involves the functioning of a complex unit of polyadenylation factors, the enzymes which catalyze the whole process. Cleavage Stimulation Factor (CstF) is a heterotrimer complex of the polyadenylation unit composed of CstF50, CstF64 and CstF77. This study highlights some aspects of the Arabidopsis thaliana homolog of CstF64, playing a vital role in RNA recognition and on the other hand in transcription termination and polyadenylation.Yeast two-hybrid interaction studies showed a novel kind of interaction of CstF64 with FIP1. It is also speculated from sub-cellular localization by agroinfiltration that CstF64 is co-localized to the nucleus in presence of CstF77, which is known to be the only CstF subunit having the nuclear localization signal in mammals. Based on these studies, a model for a sub-complex involving CstF64, CstF77 and FIP1 may be built. The ramifications of this model will be presented and discussed.

References:
Li Q, Hunt AG(1997)The Polyadenylation of RNA in Plants.Plant Physiol.115:321-325

Takagaki Y, Manley JL(2000)Complex Protein Interactions within the Human Polyadenylation Machinery Identify a Novel Component. Mol.Cell.Biol.20:1515-1525

Keywords: Polyadenylation, Cleavage Stimulation Factor (CstF), Yeast two-hybrid and Agroinfiltration

2. Identification and characterization of splicing factors in C. elegans

Daipayan Banerjee (Biology and University of Kentucky), Jim Lund (Biology and University of Kentucky), Brian Rymond (Biology and University of Kentucky)

Abstract:
Pre-messenger mRNAs are often alternatively spliced to give rise to multiple mRNA isoforms thus increasing proteome diversity. Tissue specific alternative mRNA splicing is essential for functionally different gene products from a single gene, and thus it is an important mechanism for cellular differentiation. Various human diseases are caused by the disruption of the splicing machinery. The molecular mechanisms by which alternative splicing is regulated appear varied and remain poorly understood.

A transgenic alternative splicing reporter gene in C.elegans has been developed by Kuroyanagi et al (Nature Methods, November 2006). This reporter derived from the egl-15 gene which encodes the sole homologue of fibroblast growth factor receptor (FGFR). Egl-15 has two mutually exclusive exons 5A and 5B correspond to a portion of the protein’s extracellular domain. To monitor the expression of mutually exclusive exons, GFP and RFP were introduced downstream of exon 5A and 5B respectively. Transgenic worms show differential expression of GFP and RFP proteins depending upon the tissue specific splice patterns.

Here this worm based system is used to investigate the function of phylogenetically conserved pre-mRNA splicing factors on a whole-animal basis. Specifically, we are interested in a group of proteins weakly associated with the U2 snRNP particle’s SF3b subunit that, in yeast, are necessary for efficient splicing of only a minor subset of yeast introns. The approach is to use selective RNAi-based degradation of selected splicing factors and then to monitor the impact of the knockdown on reporter gene expression and pre-mRNA splicing. Based on RNAi knockdowns in a number of tissue culture systems, I hypothesize that knockdowns of the yeast splicing factors in C. elegans will alter the pattern of splicing. This approach will provide the first experimental data for activity in splicing for some of these gene products and may provide insight into how diminished levels selective components of the splicing apparatus influence splice site choice.

Preliminary data are encouraging as clear changes in reporter gene activity occur with targeted knockdown of selective splicing factors. Furthermore, the precise differences in GFP and RFP expression that occur in response to RNAi addition appear to vary with the specific splicing factor targeted. Future studies will employ bioinformatic approaches to investigate genome-wide changes in splice site selection after knockdown of specific alternative splicing factors and core components of the spliceosome.

Keywords: splicing factor, Celegans

3. Generation of a new mouse model to help determine the therapeutic time-point for SMN replacement therapies: an update

Thomas Bebee (The Center for Childhood Cancer, Columbus Children’s Research Institute and the Department of Pediatrics, The Ohio State University School of Medicine and Public Health, Columbus, Ohio), Jordan Gladman (The Center for Childhood Cancer, Columbus Children’s Research Institute and the Department of Pediatrics, The Ohio State University School of Medicine and Public Health, Columbus, Ohio), Dawn S. Chandler (The Center for Childhood Cancer, Columbus Children’s Research Institute and the Department of Pediatrics, The Ohio State University School of Medicine and Public Health, Columbus, Ohio)

Abstract:
Proximal Spinal Muscular Atrophy (SMA), a leading genetic cause of infant mortality in humans, is caused by deletion or mutation of the survival of motor neuron gene-1 (SMN1). Homozygous deletion of the homologous gene (SMN) in mouse models leads to early embryonic lethality, arguing the necessity of SMN during early development. Humans carry a nearly identical SMN2 gene that is capable of rescuing the embryonic lethality and disease phenotype in mouse models, in a dose dependent manner. The SMN2 gene differs from SMN1 primarily in a C>T point mutation in the exon splicing enhancer (ESE) of exon 7, leading to exon 7 skipping and a truncated non-functional protein. A fraction of SMN2 transcripts include exon 7, therefore, correction of SMN2 splicing to produce increased exon 7 inclusion is an intriguing target for SMA therapies. However, the timing of SMN replacement therapies will be crucial. A temporally inducible SMN-transgenic mouse when placed in the SMA disease background will allow for the determination of this window. To achieve an inducible SMN-transgenic mouse we will place SMN expression under the control of tamoxifen inducible Cre recombination, allowing for controlled expression of SMN at varying time points during development. In our transgene construct, both drug resistance and reporter gene expression are expressed prior to Cre recombination, whereas, SMN expression is permitted only after Cre recombination. The non-recombined transgene construct was electroporated into ES cells. We screened the resultant ES cells for adequate expression by staining for the reporter gene and for a single integration event by Southern Blot. Three selected ES cell lines were then electroporated with a plasmid expressing Cre recombinase, and expression of the transgene and Cre specific recombination were assessed by PCR. These lines have been injected into Mouse blastocysts and chimeras from these lines are being tested for germline transmission.

Keywords: Spinal Muscular Atrophy, Survival Motor Neuron, Mouse Model

4. Thermodynamic characterization of e. coli manganese superoxide dismutase binding to single- and double-stranded polynucleic acids

Lana Bengez (Chemistry, John Carroll University), Iteen Cheng (Chemistry, John Carroll University), Angela C. Smolik (Technology Transfer, Case Western Reserve University), David P. Mascotti (Chemistry, John Carroll University)

Abstract:
Bacterial manganese superoxide dismutase (MnSOD) has been shown to localize to the chromosomal portion of the cell and impart protection from ionizing radiation to DNA. The binding affinity of bacterial MnSOD to non-sequence specific double stranded oligomeric DNA has been quantitated previously by nitrocellulose filter binding and gel shift assays. We have examined the equilibrium binding of E. coli MnSOD containing tryptophan to poly(U), poly(A), poly(C), poly(dU) and double-stranded (ds) DNA. Equilibrium association constants, Kobs, measured by monitoring tryptophan fluorescence quenching, were examined as functions of monovalent salt (MX) concentration and type, as well as temperature, from which deltaG°obs and deltaH°obs were determined. The polynucleotides bind to MnSOD in the following affinity hierarchy, poly(dU)>poly(U)>dsDNA>poly(A)>poly(C). For each polynucleotide, Kobs decreases with increasing [K+]. For polyU, polyA and polyC the values of deltaHobs become less favorable with increasing [K+]; therefore, the salt concentration dependence of deltaG°obs has contributions from entropic and enthalpic origins such that dlogKobs/dlog[K+] is less negative than if it were a simple electrostatic binding event.

Keywords: MnSOD, protein-RNA interactions, thermodynamics

5. Monitoring pre-mRNA Splicing by Single-Molecule Fluorescence Resonance Energy Transfer

Mario R. Blanco (Uniersity of Michigan), Mark Ditzler, Mona Wood, Jesse Sinanan (University of Michigan), Nils Walter (University of Michigan), John Abelson (University of California San Francisco)

Abstract:
The spliceosome is composed of 5 ribonucleoproteins (RNPs) which contain a total of over 100 different proteins, and 5 small nuclear RNAs (snRNAs). By acting in an ATP dependent manner, this complex is able to precisely excise diverse introns from their precursor-mRNAs (pre-mRNAs) to give mature mRNA products. Although the spliceosome has been studied extensively, there is still little known about the timing, coordination and magnitude of pre-mRNA conformational changes upon spliceosome assembly and splicing. Ensemble techniques have been unable to provide detailed kinetic information about the complicated series of events associated with splicing, and thus we chose to apply single-molecule Fluorescence Resonance Energy Transfer (sm-FRET) to a small yeast intron to gain better insight into these processes.
In our system, a pre-mRNA substrate encoding the yeast ubiquitin-conjugating protein 4 (UBC4) is labeled with Cy3 Donor (D) fluorophore in the 5’-exon and Cy5 Acceptor (A) fluorophore in the 3’-exon. The FRET efficiency between fluorophores is thus related to the distance between the two exons, allowing us to monitor the prevalence of distinct pre-mRNA conformations and transitions between them during assembly and catalytic action of the spliceosome in yeast cell extract. Our data in an ATP deficient yeast cell extract show that our pre-mRNA exhibits several low, interchanging FRET states. Upon addition of ATP we observe a clear change in mRNA structural dynamics toward higher FRET efficiencies, indicating ATP-dependent spliceosomal activity.

Keywords: splicing, mRNA, FRET

6. Relative Quantitation of Isotopically Labeled RNAs Using Data Dependent LC-MS/MS

Colette M. Castleberry (Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati), Rachel M. Selby (Department of Chemistry, Rose-Hulman Institute of Technology), Larry Sallans (Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati), Patrick A. Limbach (Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati)

Abstract:
New tools are needed to simultaneously quantify and sequence ribonucleic acids (RNAs) to identify changes in expression levels and post-transcriptional modifications. Our lab previously developed a MALDI-based method to perform relative quantification on RNAs using an enzyme-mediated isotope labeling approach. Isotope labeling is made possible during the enzymatic digestion of intact RNAs in the presence of H218O. Control samples are similarly labeled during digestion in H216O. Decreasing sample size and increasing sample complexity both pose problems for the MALDI-based method that may be solved by switching to LC-MS. To implement this approach, digestions are combined prior to analysis. Standard oligonucleotide buffers are used for HPLC with triethylamine and 1,1,1,3,3,3-hexafluoroisopropanol as ion-pairing agents. Data-dependent MS/MS provides sequence information for RNA identification. We have determined the collision energies and mass ranges appropriate for the instrument; current investigations to improve sequencing includes building library databases for CID fragments and broadening the parent ion window to pass both the 16O and 18O labeled oligonucleotides. We have found that the presence of the isotopic label does not affect oligonucleotide retention time; thus mixtures of 16O and 18O labeled oligonucleotides of the same sequence will co-elute. The relative difference in the two samples can be accurately determined over one order of magnitude. The back-exchange of 18O labeled products to 16O labeled products was also investigated to increase the accuracy and precision of this tool for both MALDI and LC-MS. The effects of time, temperature, and pH on the digestion products were studied as a possible causes of back-exchange. We anticipate that this labeling approach, in combination with RNA sequencing, will provide a powerful technique for the simultaneous quantification and identification of RNAs at throughput levels significantly greater than presently available using standard biochemical methods.

Keywords: Mass Spectrometry, High Performance Liquid Chromatography, Quantitation

7. Strategy to learn the function of the tRNA retrograde pathway via polysome RNAs and microarray analyses

Hui-Yi Chu (MCDB, the Ohio State University), Anita K Hopper (Department of Molecular Genetics, the Ohio State University)

Abstract:
In eukaryotic cells, tRNAs are transcribed in the nucleus and previously were thought to be unidirectly transported to the cytoplasm for function in protein synthesis. However, our recent studies showed that cytoplasmic tRNAs accumulate in the nucleus following amino acid deprivation in yeast and rat hepatoma cells (Shaheen and Hopper, 2005; Shaheen et al, 2007). Upon re-feeding, cytoplasmic tRNAs accumulated in the nucleus are re-exported to the cytoplasm. Current data indicate that tRNA movement includes three steps: 1) primary export of partially matured tRNA; 2) retrograde movement of tRNA into nucleus; 3) re-export of fully mature tRNA to the cytoplasm. At least three members of the β-importin family participate in tRNA subcellular dynamics. Los1 functions in step 1, Mtr10 in step 2, and Msn 5 in step 3. However, it remains unclear what the ultimate cellular consequences are of the tRNA nuclear accumulation. We propose that the retrograde process helps to regulate protein synthesis in response to nutrient deprivation. To test this hypothesis, our strategy is to monitor changes in mRNAs associated with polysomes. Therefore, we are investigating changes of protein synthesis by analyzing polysome profiles and by microarray analysis using polysomal mRNAs isolated from mtr10 and msn5 mutants grown in the presence or absence of amino acids.

Keywords: tRNA, transport, polysomes

8. NMR Structure Predictions of Hexamer and Tetramer Oligo (C)

Jenee D. Cyran (Department of Chemistry, Allegheny College), Jason Tubbs (Department of Chemistry, University of Rochester), Douglas H. Turner (Department of Chemistry, University of Rochester), Marty Serra (Department of Chemistry, Allegheny College)

Abstract:
Various two-dimensional proton NMR experiments have been completed to determine the structure of the hexamer r(C6) and tetramer r(C4) . One-dimensional variable temperature experiments determined the optimum temperature for two-dimensional experiments; the temperature with the fewest overlapping peaks is 25oC. Two-dimensional experiments were also done at 0oC to lower proton exchange. The NOESY experiments allow the order of nucleotides in r(C6) to begin to be determined from the NOESY walk. The relative distance between H6 to H5, H5 to H1’, and H6 to H1’ can be observed in the 2D NOESY experiments. These spectra illustrated that H5 appears be closer to H6 than it is to it’s H1’ in C6. The distance between H6 and H5 is known to be 2.4 Ao, and from the relative size of the cross peaks is found to be the shortest distance between H’s of the three examples. These results differ from the previous study (1) of a poly(C) which showed the H5 to H1’ to be the strongest cross peak suggesting that the H5 to H1’ distance less than 2.4 Ao. Molecular modeling of the hexamer C will be performed once the NOESY walk is finished and the NOE cross-peak volumes are computed. Future projects will help determine the solution structure of single stranded hexamer and tetramer C.

Keywords: NMR, Hexamer C and Tetramer C, Oligo (C)

9. Why is the yeast splicing endonuclease complex located in the cytoplasm and not in the nucleus?

Nripesh Dhungel (Molecular Genetics, The Ohio State University), Anita K. Hopper (Molecular Genetics, The Ohio State University)

Abstract:
Pre-tRNA splicing is a process that is critical to the viability of most eukaryotic cells. In vertebrates and yeast, the tRNA splicing endonuclease that cleaves the exon-intron junctions of pre-tRNA is composed of four proteins; Sen2p, Sen15p, Sen34p, and Sen54p. In yeast, splicing of pre-tRNAs occurs in the cytoplasm at the surface of the mitochondria (Yoshihisa et al., 2003), in contrast to mammalian pre-tRNA splicing, which occurs within the nucleus. The different location of pre-tRNA splicing machinery in yeast versus vertebrate cells raises the question of why this arrangement is maintained. My work aims to elucidate the spatial roles of the splicing endonuclease complex by directing the localization of the yeast pre-tRNA splicing machinery to the nucleus. To accomplish this, I have conjugated each protein of the splicing machinery to a nuclear localization signal and two GFP molecules. Thus far, we possess the constructs necessary to visualize different proteins of the splicing complex and we report nuclear localization of individual proteins of the machinery. Our next goal is to test that the proteins of the splicing machinery are retained in the nucleus and that they are in fact catalytically active. The ultimate goal will be to determine the physiological consequences of yeast with defective cytosolic splicing machinery and a functional complex in the nucleus.

References:
Yoshihisa T, Yunoki-Esaki K, Ohsima C, Tanaka N, Endo T.(2003). Possibility of cytoplasmic pre-tRNA splicing: the yeast tRNA splicing endonuclease mainly localizes on the mitochondria. Molecular Biology of the Cell 14,3266-3279.

Keywords: mitochondria, pre-tRNA, splicing endonuclease complex

10. Investigating the Physical Origin of Molecular Heterogeneity in the Hairpin Ribozyme

Mark A. Ditzler (Biophysics University of Michigan), David Rueda (Chemistry Wayne State University), Jingjie Mo, Kristina Hakansson, Nils G. Walter (Chemistry University of Michigan)

Abstract:
Single-molecule techniques provide unique insights into the nature of biologically important macromolecules by revealing both s hort-lived intermediates and weakly populated sub-populations not observed in ensemble measurements. Consequently, molecular heterogeneity has been detected for a variety of systems at the single molecule level. For the hairpin ribozyme, analysis of single-molecule fluorescen ce resonance energy transfer (FRET) time trajectories reveals the presence of persistent heterogeneity in global (un)docking dynamics (1, 2). At least four folding sub-populations have been detected in the minimal hairpin ribozyme. The folding sub-populations are distinguishe d from each other based on their undocking kinetics. The different undocking behaviors persist for individual molecules over long periods of time and through multiple cycles of docking and undocking, giving rise to a so-called "memory effect"(1).
In this work, we characterize the physical origin of the heterogeneous dynamics of the hairpin ribozyme by correlating the heterogeneity observed at the single-molecule level with ensemble measurements. We used native polyacrylamide gel electrophoresis (PAGE) to separate two previously observed folding populations of the hairpin ribozyme (3). Single-molecule and time-resolved ensemble FRET reveal that the gel mobility of the two populations is primarily determined by the molecular heterogeneity observed at the single-molecule level. Chemical and enzymatic foot-printing reveals a shared secondary structure between the two populations. Additionally, native PAGE assays demonstrate that denaturation/refolding alone is not sufficient to inter-convert the two populations, indicating that the ribozyme may be covalently modified. We have applied fourier transform ion cyclotron resonance mass spectrometry to search for covalent modifications of the ribozyme. Our mass spectrometry results do not support the presence of covalent modifications that change the mass. Therefore, our current results are most consistent with models in which the "memory effect" either arises from a mass-neutral covalent modification or from an extraordinarily stable alternate tertiary fold.

References:
1. Zhuang, X., et al., Science, 2002. 296(5572): p. 1473-6.
2. Rueda, D., et al., Proc Natl Acad Sci U S A, 2004. 101(27): p. 10066-71.
3. Pinard, R., et al., Embo J, 2001. 20(22): p. 6434-42.

Keywords: FRET, Single-molecule, Kinetic Trap

11. Binding studies of a peptide with the A-site rRNA

Anne-Cecile E. Duc (Wayne State University), Mei Li (Wayne State University), Christine S. Chow (Wayne State University)

Abstract:
The increasing number of resistant bacteria to the existing antibiotic panoply is of great concern. Among these antibiotics, almost half are interfering with the protein synthesis machinery, whose core is the ribosome. Within its two subunits, the ribosome harbors several sites targeted by antibiotics, one of them being the A site. The A-site rRNA, located on helix 44 of the small subunit 16 S rRNA, is involved in the universal decoding phenomenon, and therefore offers few possibilities for mutations. In our research, phage display was previously used to identify a peptide that binds to the 27-nucleotide A-site RNA model. The identification of the key amino acids in the binding to the RNA was determined by performing single and double alanine screening. The variant peptides were analyzed using biophysical methods such as electrospray-ionization mass spectrometry, enzymatic footprinting and circular dichroism. These studies give some insights about the affinity and specificity of the peptide variants with the E. coli A-site RNA. Understanding these interactions will lead us to modify the peptide to enhance its affinity and specificity, therefore its activity.

References:
Steitz. T. A., On the structural basis of peptide-bond formation and antibiotic resistance from atomic structures of the large ribosomal subunit. FEBS Letters 2005, 579, (4), 955-958
Fourmy D., R. M. I., Blanchard S. C., Puglisi J. D., Structure of the A Site of Escherichia coli 16 S Ribosomal RNA Complexed with an Aminoglycoside Antibiotic. Science 1996, 274, (5291), 1367-1371

Keywords: RNA, peptide, Ribosome

12. A model for the flexibility of double-stranded DNA incorporating local melting

Robert A. Forties (Department of Physics, The Ohio State University), Ralf Bundschuh (Department of Physics, The RNA Group, Biophysics Graduate Program, The Ohio State University), Michael Poirier (Department of Physics, Biophysics Graduate Program, Ohio State Biochemistry Program, The Ohio State University)

Abstract:
Many biological processes, including gene regulation, binding of transcription factors,and DNA packaging, require the formation of DNA loops. We posit that melted segments of double­ stranded DNA (dsDNA) are more flexible than closed dsDNA, and therefore that dsDNA constrained to a loop may melt locally to reduce the overall bending energy for the loop. We computationally predict J factors, which are proportional to the probability of cyclization, over a range of temperatures. Our model predicts that cyclization is more favorable at higher temperatures, as more local melting will occur. We also predict that DNA molecules containing segments with low melting temperatures will cyclize more readily than predicted by the standard worm-like chain model.

We measure J factors in cyclization experiments for a 200 basepair (bp) fragment of lambda DNA and two 116 bp sequences. One 116 bp sequence is designed to melt more easily, and therefore cyclize more readily than the other. We determine J factors by measuring the concentrations of fluorescently labeled ligation products visualized on polyacrylamide gels. The measured temperature and sequence dependence of J factors is found to be in agreement with our model predictions using reasonable values for the flexibility of melted segments of dsDNA.

Keywords: DNA cyclization, DNA bending, DNA melting

13. SMN2 exon 7 knock-in: A new spinal muscular atrophy mouse model.

Jordan T. Gladman (The Center for Childhood Cancer, Columbus Children’s Research Institute, The Department of Pediatrics, The Ohio State University.), Thomas Bebee (The Center for Childhood Cancer, Columbus Children’s Research Institute, The Department of Pediatrics, The Ohio State University.), Dawn S. Chandler (The Center for Childhood Cancer, Columbus Children’s Research Institute, The Department of Pediatrics, The Ohio State University.)

Abstract:
Proximal spinal muscular atrophy (SMA) is caused by loss of the survival motor neuron-1 (SMN1) gene leaving only the SMN2 gene to produce the essential SMN protein. SMN2 varies from SMN1 at a single nucleotide in exon 7 which leads to ineffective processing of the RNA by the cellular splicing machinery causing skipping of exon 7, and the generation of a non-functional protein. We hypothesize that the introduction of the human SMN2 mutation in the mouse Smn gene will more closely resembles the human condition in which exon 7 skipping occurs. In order to test the mouse Smn gene’s ability to skip exon 7 in the presence of the human SMN2 point mutation, we constructed a series of Smn mini-genes directly amplified from the mouse 129Sv/Ev genome. Using these mini-genes in transfection experiments we find that transcripts containing the native sequences yield mRNA containing all three exons while the mini-gene containing the exon 7 point mutation predominantly yields the exon 7 skipped mRNA in both human and mouse cell lines. Additionally we have shown up to 70% skipping of exon 7 using both mouse and human mini-genes. These experiments support the idea that a point mutation in exon 7 of the mouse gene will affect SMN splicing in the mouse tissues, as in humans. To generate a mouse harboring the SMN C>T mutation in exon 7 we designed a targeting construct containing the C>T point mutation. A single targeted ES cell line that has undergone correct homologous recombination was used to generate a germline population mice carrying the SMN2 exon 7 C>T point mutation in their Smn gene. We believe that the resultant mouse will more accurately recapitulate the SMA splicing phenotype observed in the human condition, and can be used to address potential therapies aimed at correcting SMN2 splicing.

Keywords: Proximal Spinal Muscular Atrophy, Survival Motor Neuron, Mouse Model

14. Structural transitions of the Bacillus subtilis glyQS T box leader RNA

Nicholas J. Green (Microbiology, Ohio State), Frank J. Grundy (Microbiology, Ohio State), Tina M. Henkin (Microbiology, Ohio State)

Abstract:
Many aminoacyl-tRNA synthetase (aaRS) genes and other amino acid related genes in Gram-positive bacteria are regulated by the T box transcription termination mechanism. In this system, the nascent leader RNA transcript senses the charging ratio of the cognate tRNA through specific base pairing interactions. Expression of an individual gene is induced by limitation for its cognate amino acid, which results in reduced charging of the cognate tRNA. Readthrough of a termination site in the leader region of the transcript and expression of the downstream gene relies on stabilization of an antiterminator helix through interactions with the uncharged tRNA, preventing the formation of a terminator helix and allowing synthesis of the full length mRNA.

Specific interactions of the anticodon and the acceptor end of the tRNA with the transcribed leader RNA have been well-characterized by genetic and biochemical methods, supporting the structural transition of the terminator helix and the proposed secondary structure model. However, less is known about the tertiary structure of the leader RNA or the functional role of other conserved RNA elements. Chemical and enzymatic mapping of the 5\' UTR of the Bacillus subtilis glyQS operon (encoding glycyl-tRNA synthetase) has revealed novel tRNA-induced changes at positions near several of these conserved regions within the leader. Mutations of the glyQS leader are discussed, with the C160U mutation shown as a specific example of a conserved nucleotide critical for the formation of the antiterminator helix, and stability of the tRNA:leader RNA complex.

References:
Grundy FJ, Henkin TM. tRNA as a positive regulator of transcription antitermination in B. subtilis. Cell. 1993 Aug 13; 74(3):475-82.

Grundy FJ, Henkin TM. The T box and S box transcription termination control systems. Front Biosci. 2003 Jan 1; 8:d20-31.

Grundy FJ, Henkin TM. Regulation of gene expression by effectors that bind to RNA. Curr Opin Microbiol. 2004 Apr; 7(2):126-31.

Grundy FJ, Moir TR, Haldeman MT, Henkin TM. Sequence requirements for terminators and antiterminators in the T box transcription antitermination system: disparity between conservation and functional requirements. Nucleic Acids Res. 2002b Apr 1; 30(7):1646-55.

Grundy FJ, Winkler WC, Henkin TM. tRNA-mediated transcription antitermination in vitro: codon-anticodon pairing independent of the ribosome. Proc Natl Acad Sci U S A. 2002a Aug 20; 99(17):11121-6.

Merino E, Yanofsky C. Transcription attenuation: a highly conserved regulatory strategy used by bacteria. Trends Genet. 2005 May; 21(5):260-4.

Rollins SM, Grundy FJ, Henkin TM. Analysis of cis-acting sequence and structural elements required for antitermination of the Bacillus subtilis tyrS gene. Mol Microbiol. 1997 Jul; 25(2):411-21.

Yousef MR, Grundy FJ, Henkin TM. Structural transitions induced by the interaction between tRNA(Gly) and the Bacillus subtilis glyQS T box leader RNA. J Mol Biol. 2005 Jun 3; 349(2):273-87.

Keywords: RNA RNA interaction, Structure, Transcription Regulation

15. Single Molecule Studies of spliceosomal snRNAs U2-U6

Zhuojun Guo (Department of Chemistry, Wayne State University), David Rueda (Department of Chemistry, Wayne State University)

Abstract:
Splicing is an essential step in the maturation of eukaryotic pre-mRNA in which intervening sequences (introns) are removed from the coding sequences (exons). The spliceosome is a dynamic assembly of five snRNAs and a large number of proteins- that catalyzes the splicing process. U2 and U6 are the only two spliceosomal snRNAs strictly required for both steps of splicing, and they form RNA complex using base pairing. Major conformational changes are expected to take place during the assembly and catalysis of the spliceosome.
Here, we have used Fluorescence Resonance Energy Transfer and Single Molecule Spectroscopy to study the structural dynamics of a protein free U2-U6 complex from S. cerevisiae. Our FRET data clearly shows a Mg2+ induced large amplitude conformation change of the U2-U6 complex. In the absence of Mg2+ helix I and the U6-ISL are in close proximity, while in the presence of Mg2+ these two helices are far from each other. Our single molecule data shows that the conformational change consists of a two-step process, with an obligatory folding intermediate. We propose that the observed conformational change corresponds to the activation process expected to occur during spliceosomal assembly and catalysis and this hypothesis is confirmed by the recent data from mutant U2-U6 complex which contains point mutations in highly conserved regions.

Keywords: spliceosome, U2-U6 complex, Single Molecule Spectroscopy

16. Missense or silent mutations in p53 exon 7 ESE result in exon skipping

Reeva Gupta (The Center for Childhood Cancer, Columbus Childrens Research Institute and the Department of Pediatrics, The Ohio State University School of Medicine and Public Health, Columbus, Ohio), Jessica Fleming (The Center for Childhood Cancer, Columbus Childrens Research Institute, Columbus, Ohio), Jill Molfenter (The Ohio State University School of Medicine and Public Health, Columbus, Ohio), Dawn S. Chandler (The Center for Childhood Cancer, Columbus Childrens Research Institute and the Department of Pediatrics, The Ohio State University School of Medicine and Public Health, Columbus, Ohio)

Abstract:
Germ line mutations in TP53 gene have been observed in Li-Fraumeni Syndrome, a familial cancer syndrome. This syndrome is characterized by a diverse set of early-onset malignancies including breast carcinoma, sarcomas and brain tumors. Germ line analysis for p53 has reported mutations between exon 5 and 9.

The most commonly observed missense mutations are within codon 248. The changes in exon 7 results in amino acid alterations from an arginine (R) to either trypothan (W) or glutamine (Q) which affect protein function. Incidentally silent mutations have been identified at the same location. ESE finder database predicted that 248 codon lies within the two overlapping SF2/ASF predicted binding sites. Interestingly, the four reported 248 missense and silent mutations were predicted to disrupt one or both the binding sites. We hypothesize that these mutations disrupt p53 normal splicing, resulting in truncated p53 protein with altered function that ultimately contributes to the cancer phenotype.

To confirm the candidate ESE function for codon 248 in exon 7 we are using minigene splicing assays. The exon 7 ESE was cloned in the SXN-13 minigene exon 2 to determine the skipping or inclusion of the exon 2. The ESE was also altered to include the four reported exon 7 mutations.

Our results indicate that the wild type p53 ESE promotes proper exon recognition while mutant ESEs give rise to transcripts in which the exon has been skipped. The most deleterious splicing mutations in the exon 7 ESE are CGG248CGT (silent), CGG248CGA (silent), CGG248TGG(R > W), CGG248CAG(R > Q).

We are currently analyzing patient cell lines which harbor these p53 ESE mutations for the presence of the altered p53 transcripts. The identification of silent p53 mutation that affect p53 splicing will help in determination of the functional role of these new spliced isoforms in the initiation of cancer.

Keywords: Exon splicing enhancers, TP53, silent or missense mutations

17. Identification of methylation sites on Thermus thermophilus ribosomal protein L11

Daisy-Malloy Hamburg (Department of Chemistry, University of Cincinnati), Anne McLachlan, Patrick A. Limbach (Department of Chemistry, University of Cincinnati), Hasan Demirci, Steven T. Gregory, Albert E. Dahlberg (Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University)

Abstract:
In this work, we use mass spectrometry to identify the methylation sites on T. thermophilus ribosomal protein L11. Protein L11, located in the 50S subunit of the ribosome, is known to be post-translationally trimethylated at several residues by a single methyltransferase, PrmA. L11, the most heavily methylated component of the bacterial translational apparatus, is universally conserved and actively participates in interactions of the ribosome with protein synthesis factors during initiation, elongation and termination phases of translation. L11 in Escherichia coli is known to be trimethylated at three positions: the á-amino group of the N-terminal amino acid and the ĺ-amino group of lysine 3 and lysine 39 [2]. MALDI-TOF MS data of L11 in T. thermophilus revealed that L11 is trimethylated at an additional fourth residue [2]. Recently, Demirci et al [3] described four structures of PrmA from T. thermophilus, revealing how PrmA can position the L11 substrate for multiple consecutive side-chain methylation reactions. Here we use LC MS/MS to deduce the location of the fourth trimethylation.

References:
[1] Dognin MJ, Wittman-Liebold B (1980) Eur J Biochem 112: 131-151.
[2] Cameron DM, Gregory ST, Thompson J, Suh MJ, Limbach PA, Dahlberg AE (2004a) J Bacteriol 186: 5819-5825.
[3] Demirci H, Gregory S, Dahlberg AE, Jogi G (2007) EMBO J 26: 567-577.

Keywords: Methylation, LC MSMS

18. Development of compounds that increase SMN2 splicing as a treatment for spinal muscular atrophy

Michelle L. Hastings (Dept. of Cell Biology and Anatomy, Chicago Medical School), Joel Berniac (Paratek Pharmaceuticals, Inc.), Ying Hsiu Liu (Cold Spring Harbor Laboratory), Paul Higgins (Paratek Pharmaceuticals, Inc), Adrian R. Krainer (Cold Spring Harbor Laboratory)

Abstract:
Spinal muscular atrophy (SMA) is a pediatric neurodegenerative disease caused by loss of the survival of motor neuron 1 (SMN1) gene. A second gene, SMN2 is nearly identical to SMN1 but cannot compensate for the loss of SMN1 because most SMN2 mRNA transcripts lack exon 7 as a result of a C to T conversion at position +6 of exon 7. Transcripts lacking exon 7 produce a truncated protein that cannot function in place of the full-length SMN protein. Correcting aberrant SMN2 exon 7 splicing is one way to increase full-length SMN protein expression from SMN2 as an approach for SMA therapy. We developed an in vitro cell-free assay to test for compounds that improve SMN2 exon 7 splicing directly. One class of compounds tested was modified tetracycline (TC) derivatives and one compound, P1033, emerged as a promising hit. P1033 treatment leads to a 6-fold increase in SMN2 exon 7 inclusion in our in vitro assay and leads to a 40% increase in SMN protein levels in SMA patient-derived fibroblasts. P1033 increases exon 7 inclusion by 74% in mice carrying the human SMN2 transgene, demonstrating the promise of this molecule as a therapeutic. Medicinal chemistry using structure-activity relationship (SAR) analysis has yielded additional TC derivatives with activity in the splicing assay. Future efforts will focus on optimizing the potency and pharmacokinetic profile of these novel derivatives to identify a pre-clinical candidate for the treatment of SMA.

Keywords: splicing, disease

19. An aminoacyl-tRNA synthetase:elongation factor complex for substrate channeling in archaeal translation

Corinne D. Hausmann (Department of Microbiology, The Ohio State University), Mette Praetorius-Ibba (Department of Radiology, The Ohio State University), Michael Ibba (Department of Microbiology, The Ohio State University)

Abstract:
Translation requires the specific attachment of amino acids to tRNAs by aminoacyl-tRNA synthetases (aaRSs) and the subsequent delivery of aminoacyl-tRNAs to the ribosome by elongation factor 1 alpha (EF-1a). Interactions between EF-1a and various aaRSs have been described in eukaryotes, but the role of these complexes remains unclear. To investigate possible interactions between EF-1a and other cellular components, a yeast two-hybrid screen was performed for the archaeon Methanothermobacter thermautotrophicus. EF-1a was found to form a stable complex with leucyl-tRNA synthetase (LeuRS; Kd = 0.7 uM). Complex formation had little effect on EF-1a activity, but increased the rate of Leu-tRNALeu synthesis 8-fold. In addition, EF-1a co-purified with the archaeal multi-synthetase complex (MSC) comprised of LeuRS, LysRS and ProRS, suggesting the existence of a larger aaRS:EF-1a complex in archaea. These interactions between EF-1a and the archaeal MSC may promote the efficiency of translation both by enhancing the aminoacylation efficiencies of the three associated aaRSs and by coupling two stages of translation: aminoacylation of cognate tRNAs and their subsequent channeling to the ribosome.

References:
Hausmann, C.D., Praetorius-Ibba, M., and Ibba, M. (2007) Nucl. Acids Res. In press.

Praetorius-Ibba, M., Hausmann, C.D., Paras, M., Rogers, T.E., and Ibba, M. (2007) J. Biol. Chem., 282, 3680-3687.

Keywords: Elongation factor 1 alpha; EF-1a, Aminoacyl-tRNA synthetase; aaRS

20. Regulation of the protein kinase PKR by perfect and defect dsRNAs

Laurie A. Heinicke (Department of Chemistry, The Pennsylvania State University), Amy Diegelman-Parente (Division of Mathematics and Natural Sciences, Altoona College The Pennsylvania State University), Subba Rao Nallagatla (Department of Chemistry, The Pennsylvania State University), Philip Bevilacqua (Department of Chemistry, The Pennsylvania State University)

Abstract:
PKR is an RNA regulated protein kinase and is part of the innate immune response. PKR is phosphorylated in the presence of certain RNA structures with downstream effects including inhibition of translation and regulation of cell differentiation. PKR is activated by perfect double-stranded RNAs (dsRNAs) greater than 33 bp, but is also activated by minimally structured single-stranded RNAs (ssRNAs) containing a 5’-triphosphate and dsRNAs with numerous imperfections. In this study, three types of RNAs were tested in PKR activation assays; perfect dsRNAs, minimally structured ssRNAs, and biologically relevant Hepatitis Delta Virus (HDV) RNAs.

Time-course activation assays have been performed to investigate the mechanism of PKR activation by perfect 79 bp RNA and minimally structured RNA. In the presence of 79 bp RNA, a 1 to 2 minute lag is observed followed by an increase of activation to 10 minutes. Activation assays in the presence of urea and at various temperatures suggest that PKR is not very stable. Numerous cleavable and non-cleavable HDV RNA constructs have been tested for PKR activation. Short HDV constructs do not activate PKR, while most longer HDV constructs containing increasingly complex secondary and tertiary structures and less than 33 consecutive base pairs do activate PKR. Interestingly, two short constructs (-54/-1 and 1/99) do not activate PKR, whereas a non-cleavable form of the combined constructs (-54/99 C75U) does activate PKR. In addition, PKR activation is inhibited by a long non-cleavable construct (-54/226 C75U) that contains a stem-loop (13 bp) connected to a long defect 94 bp rod. Regulation of PKR by intermediate HDV RNA transcripts has biological implications due to the in vivo dynamics of RNA folding during transcription. These findings may be useful in identifying general inhibitory and activating elements in other viral RNAs.

Keywords: PKR

21. Mammalian Hu protein family members have non-redundant functions as splicing suppressors

Melissa N. Hinman (Department of Genetics, Case Western Reserve University), Hui Zhu (Genomic Medicine Institute, Cleveland Clinic Foundation), Robert A. Hasman, Kavita Praveen (Department of Genetics, Case Western Reserve University), Hua Lou (Department of Genetics, Center for RNA Molecular Biology, Case Comprehensive Cancer Center, Case Western Reserve University)

Abstract:
Mammalian Hu proteins are a family of RNA-binding proteins that interact with AU-rich elements. Three of the four known family members, HuB, HuC, and HuD, are neuron-specific, while HuA (HuR in humans) is widely expressed. Hu proteins regulate mRNA stability and translation, and play an important role in neuronal differentiation. Our recent studies demonstrate a novel role for Hu proteins as splicing regulators that promote skipping of exon 23a of the neurofibromatosis type I (NF1) pre-mRNA.
Despite a high degree of sequence homology among the four members of the Hu family, it is predicted that they have distinct functional roles based on their expression patterns during development and in mature neurons. To determine if individual Hu members have differential effects on alternative splicing, we co-transfected cells with each member and a NF1 splicing reporter. Interestingly, individual Hu protein family members exhibit diverse potencies as splicing suppressors: HuR and mHuC strongly suppress NF1 exon 23a inclusion, hHuD has a moderate effect, and, most interestingly, mHuB has little effect. Immunofluorescence studies indicate that differential cellular localization is not responsible for the diverse splicing suppressor potencies of Hu family members.
To determine the amino acid basis of the differential effect of Hu proteins, we compared mHuB and mHuC sequences. Although the three RRM domains are highly homologous, the N-terminus and the hinge domain between RRM2 and RRM3 show great variability. When the N-terminal amino acids between the two proteins were swapped, the resulting fusion proteins acted like the parental proteins, indicating that the differential effect on splicing is not dictated by the sequence on the N-terminus of these proteins. We hypothesize that sequence variability in the hinge domain is responsible for the observed differential splicing suppressor strength. Our future experiments will focus on studying the role of the hinge domain in splicing regulation.

Keywords: Hu proteins, Alternative splicing, Neurofibromatosis type I

22. Conformational changes in B. subtilis RNase P holoenzyme associated with substrate binding measured by transient fluorescence techniques

John Hsieh (Department of Chemistry, University of Michigan), Daina Zeng (Department of Chemistry, University of Michigan), Carol A. Fierke (Departments of Chemistry and Biological Chemistry, University of Michigan)

Abstract:
Ribonuclease P (RNase P) catalyzes the 5’ maturation of precursor tRNA, generating a mature tRNA and a short 5’ leader RNA. The Bactillus subtilis RNase P contains a catalytically active RNA component and a small protein cofactor known as P protein, and both components contribute to molecular recognition. To further evaluate the catalytic mechanism, we have developed a complete kinetic scheme for the B. subtilis RNase P using transient kinetic techniques. We measured the association and dissociation rate constants by mixing RNase P with pre-tRNA substrates containing a fluorescent label at either the 5’ leader or the 3’ tRNA moiety and measured the time-dependent changes in fluorescence by stopped-flow techniques. We also measured the chemical cleavage rate constant using chemical quench flow techniques. Under single-turnover conditions ([E] >> [S]), we have observed two phases in the binding kinetics: a rapid, diffusion-controlled association step followed by a step with hyperbolic dependence on holoenzyme concentration where the limiting rate constant is faster than the cleavage rate constant. This indicates that a conformational change in the holoenzyme-substrate complex precedes the cleavage step. This step is also facilitated by the addition of divalent cations. In addition, the dissociation rate constants from the E•tRNA•leader complex are identical for both tRNA and the 5’ leader sequence suggesting that a rate-limiting conformational change step also precedes release of both products. This is different than the kinetic scheme for the PRNA-catalyzed reaction where the 5’ leader dissociates before the mature tRNA. This difference is likely due to the interaction of the pre-tRNA leader with the P protein in the holoenzyme.

Keywords: RNase P, transient kinetics, enzyme mechanism

23. Connections between starvation, translation, and tRNA movement?

Rebecca Hurto (Molecular Genetics, OSU), Mike Whitney (Penn State University), Abul K. Azad, Anita K Hopper (Molecular Genetics, OSU)

Abstract:
In Saccharomyces cerevisiae, tRNAs are not only exported from the nucleus to the cytoplasm, but also undergo retrograde movement that returns tRNAs to the nucleus. Cells harboring conditional defects in tRNA aminoacylation or starved for various nutrients accumulate tRNAs, which were previously located in the cytoplasm, with in their nuclei. To uncover novel factors important for tRNA nuclear export and/or re-export, a screen for muliticopy suppressors of temperature sensitive growth inhibition caused by the tys1-1 mutation was conducted and Sbp1 and Tat1 were identified. Tat1 is an amino acid permerase that imports amino acids from the media into the cytoplasm. The increased intracellular pools of tyrosine, resulting from TAT1 overexpression, may suppress the growth inhibition and tRNA nuclear accumulation caused by the tys1-1 mutation by increasing the rate of aminoacylation. Sbp1 is a cytoplasmic protein of unknown function that has a nuclei acid binding motif and is involved in glucose starvation induced P-body formation and translational repression. Overexpression of Sbp1 has been previously shown to cause translational repression independent of starvation (Segal et al., 2006), while deletion of SBP1 enhances the defect in translation repression caused by deletion of DHH1 or PAT1 (Dhh1 and Pat1 function in parallel to promote P-body formation; Segal et al., 2007). Additionally, dhh1pat1 cells do not accumulate tRNA with nuclei in response to glucose or amino acid starvation. One possible explanation undergoing testing is that overexpression of Sbp1 allows translation to continue in cells containing the tys1-1 mutation by altering the rate of translation initiation to match the availability of aminoacylated tRNATyr, thereby preventing stress signaling caused by impaired translation.

Keywords: tRNA, translation, nutrient starvation

24. tRNA-dependent Switch in Editing Mechanisms Used by Class II ProRS

Michael Ignatov (Dept. of Chemistry, Ohio State University), Kathryn Splan (Dept. of Chemistry, University of Minnesota), Karin Musier-Forsyth (Depts. of Chemistry and Biochemistry, Ohio State University)

Abstract:
Aminoacyl-tRNA synthetases are essential enzymes, which charge amino acids to their cognate tRNAs in a two-step reaction that involves aminoacyl-adenylate formation followed by aminoacyl-tRNA synthesis. Unique amino acid recognition can generally not be achieved and some amino acids are mischarged by synthetases. To achieve a high degree of fidelity during protein synthesis, an editing mechanism is required to clear mischarged tRNAs. In the so-called double-sieve mechanism, amino acids similar in size and charge can be activated and transferred to the tRNA at the synthetic active site of the enzyme and are then transferred to the editing domain for hydrolysis of non-cognate aminoacyl-tRNA. Pre- and post-transfer editing pathways are responsible for clearing of non-cognate adenylates and aminoacyl-tRNAs, respectively. Here we present a study of the editing mechanisms used by ProRSs from all three Kingdoms of life to clear misactivated Ala-AMP and mischarged Ala-tRNAPro. In the presence of tRNA, post-transfer editing is the dominant pathway of editing. In contrast, in the absence of tRNA, pre-transfer editing of misactivated Ala-AMP occurs primarily through enzymatic hydrolysis at the synthetic active site. In the case of human ProRS, formation of Ala-AMP is rate limiting compared to its hydrolysis. In the case of E. coli and Methanococcus jannaschii ProRS, hydrolysis of the adenylate is the rate-limiting step.

Keywords: aminoacyl-tRNA synthetase, editing mechanism, ProRS

25. Functional analysis of BORG, a large non coding RNA

Fereshte Jahaniani Kenari (RNA center, Case Western Reserve University), Saba Valadkhan (RNA center, Case Western Reserve University)

Abstract:
Non-coding RNAs (ncRNAs) play significant roles in many crucial aspects of cellular function such as RNA processing, mRNA stability, translation, protein stability and secretion. Despite the rapid progress in our understanding of small non-coding RNAs\' roles, the mechanism of function of larger non-coding RNAs is almost totally unknown. To gain insight into the function of these RNAs we are analyzing the function of BMP/OP- responsive gene (BORG), a 2846 nt non-coding RNA which was originally described in a screen for genes that change their expression in response to bone morphogenic protein 2 (BMP2) in mice.

The 15108 nt-long BORG pre-mRNA contains three exons and two introns, and it also contains a typical poly A signal at the 3’ end. After splicing, the mature message is 2846 nt long and contains regions with homology to B4a and mouse LTR repeat elements. We first ensured that BORG is indeed a non-coding RNA by phylogenetic analysis of the short (<300 bp long) ORFs found in its sequence. All predicted ORFs contained mutations affecting the initiation codon, early frameshift-causing mutations, or early nonsense mutations when mouse and rat BORGs were compared, indicating that the BORG transcript does not have protein-coding potential. We next studied the expression profile of BORG in several embryonic and adult mouse tissue by semi quantitative RT-PCR. BORG has a tissue specific expression pattern, with high expression levels detected in neural tissues, kidney, testis (in adults only) and cardiac tissue (in fetuses only). Efforts are underway to use in situ hybridization to determine the subcellular and organ-level localization of this non-coding RNA.

Borg is located on chromosome 15 in mouse genome, between BAALC (Brain and Acute Leukemia, cytoplasmic) and ATPase, H+ transporting, lysosomal V1 subunit C1 genes. shRNA targeting of BORG in pro-myeloblastic C2C12 mouse cell line does not affect the expression of a number of house keeping genes tested, however, it results in a dramatic repression of the neighboring BAALC gene. BAALC is expressed in neuroectoderm-derived tissues and is highly conserved among mammals. The other neighboring gene of BORG, the ATPase, does not show a significant effect. These data suggest that this non-coding RNA might function, at least partially, through regulation of expression of its neighboring genes, a feature observed in a number of non-coding RNAs. Further, the expression level of BORG shows an increase after serum starvation and heat shock, as determined by semi-quantitative RT-PCR.

To gain more insights into the function of BORG, We aim to discover whether there are human homologue of the mouse BORG gene. Preliminary studies show that the conserved region of this gene is expressed in a tissue specific manner in a few human tissues. Therefore we plan to look for the expression profile of this gene in different human tissues and to also to determine if there is a role in brain development in human for this RNA.

Keywords: large ncRNA

26. Characterization of the functional group in human U6/U2 complex

Yasaman Jaladat (RNA Center, Case Western Reserve University)

Abstract:
We have shown that human spliceosomal snRNAs U6 & U2- can perform a reaction similar to the splicing reaction. To gain insight into the splicing reaction, we have embarked on a detailed functional characterization of this minimal U6&U2 “Spliceosome�. Specifically, we are interested in defining the functional groups needed for catalysis, including those important in Mg++ coordination.

To this end, we use Nucleotide Analogue Interference Mapping to determine the functional groups needed for catalytic activity in this minimal system. To reach these means, we have tightened the binding of U6&U2 and one of the splicing substrates to ensure the formed product remains associated to the U6/U2 that has catalyzed its formation. The other splicing substrate has a biotin at its 5' end, which will allow us to separate reactive U6/U2 in complex with the product from unreactive U6/U2s. Further, in parallel experiments, we are in the process of determining the metal binding sites in the U6U2 complex using Terbium cleavage assay.

Taken together, the above experiments will allow us to define several functional aspects of the active site of the human spliceosome.

Keywords: Minimal Splicing Reaction, U6/U2, Metal Ions

27. RNA duplex unwinding by the DEAD-box protein Ded1p involves multiple enzyme units that utilize ATP differentially

Huijue Jia (Department of Biochemistry, and Center for RNA Molecular Biology, Case Western Reserve University, Cleveland, OH), Fei Liu (Department of Physics, Case Western Reserve University, Cleveland, OH), Quansheng Yang (Department of Biochemistry, and Center for RNA Molecular Biology, Case Western Reserve University, Cleveland, OH), Eckhard Jankowsky (Department of Biochemistry, Center for RNA Molecular Biology, and Department of Physics, Case Western Reserve University, Cleveland, OH)

Abstract:
In virtually all aspects of RNA metabolism, DEAD-box proteins carry out a range of ATP-dependent RNA and RNP remodeling reactions, including unwinding of RNA duplexes. Duplex unwinding by DEAD-box proteins differs from the translocation-based mechanism employed by structurally related canonical DNA and viral RNA helicases. Local strand separation, rather than translocation, underlies the unwinding activity of DEAD-box proteins. In this unwinding mode the enzyme is first loaded directly onto the duplex by a single stranded substrate region. Subsequently, the enzyme actively opens only part of the duplex locally around its binding site, which is sufficient to promote the rapid separation of duplexes shorter than approximately one and a half helical turns.

To understand this novel helicase mechanism in more detail, we have dissected individual steps of the strand separation process for the DEAD-box protein Ded1p from Saccharomyces cerevisiae. Here we show that the single stranded substrate region serves solely to load the enzyme on the duplex, with no effect on the actual unwinding. We further show that multiple protomers of Ded1p bind the single stranded substrate region and that binding site size for individual Ded1p protomer comprises approximately 10 nucleotides. Finally, we demonstrate that Ded1p units on the single stranded substrate region do not directly participate in the unwinding process but serve to load additional Ded1p units onto the duplex region, and that only ATP hydrolysis from Ded1p units binding directly to the duplex is required for unwinding. Our data reveal a complex unwinding process for the DEAD-box protein Ded1p involving multiple enzyme units, which utilize ATP differentially. This intricate unwinding mechanism may be advantageous for accommodating the many distinct RNA substrates that Ded1p may encounter during its various cellular functions.

Keywords: DEAD-box protein, RNA duplex unwinding, oligomerization

28. Insights into the chaperone function of HIV-1 Gag protein

Christopher Jones (Departments of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210), Siddhartha Datta (Retrovirus Assembly Section, HIV Drug Resistance Program, National Cancer Institute, Frederick, MD 21702), Alan Rein (Retrovirus Assembly Section, HIV Drug Resistance Program, National Cancer Institute, Frederick, MD 21702), Karin Musier-Forsyth (Departments of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210)

Abstract:
Human immunodeficiency virus type-1 (HIV-1) recruits and packages host cell tRNALys into virions for use as a primer in reverse transcription. The specific annealing of tRNALys,3 to the Primer Binding Site (PBS) on the HIV-1 genome is facilitated by HIV-1 Gag polyprotein’s potent nucleic acid chaperone activity, which is believed to reside primarily in the 55-amino acid nucleocapsid protein (NC) domain. In general, nucleic acid chaperone activity promotes the rearrangement of RNA/DNA to a thermodynamically more stable conformation. Both full-length Gag and the NC domain alone anneal the tRNA primer to the PBS in vitro, but Gag displays more potent chaperone activity than NC. Exactly how Gag’s other major structural domains, capsid (CA) and matrix (MA), contribute to its overall annealing ability is poorly understood. In this work, a series of Gag variants was constructed and tested for their ability to bind, aggregate, and anneal tRNALys,3 to the viral PBS. By mutating and deleting MA, CA, and NC domains of Gag, insights were gained into HIV-1 Gag protein’s remarkable chaperone function.

Keywords: HIV, Gag, tRNA

29. Comprehensive thermodynamic parameter analysis of 3' double overhangs on terminal wobble base pairs

Laura Jones (Chemistry Department, Allegheny College), Stacy Miller (Chemistry Department, Allegheny College), Karen Giovanetti (Chemistry Department, Allegheny College), Dr. Martin J Serra (Chemistry Department, Allegheny College)

Abstract:
Comprehensive thermodynamic parameter analysis of 3' double overhangs on terminal wobble base pairs |Thermodynamic parameters are reported for 32 self-complimentary RNA duplexes in 1 M NaCl. Twenty-four sequences contain wobble terminal base pairs (GU) with 3'double-nucleotide overhangs, mimicking the structures of short interfering RNA's (siRNA) and microRNAs (miRNA), and eight sequences contain 3' single-nucleotide overhangs. Results from this study allow for the development of a refined nearest-neighbor model to predict the influence of 3' double overhangs on wobble terminal base pairs on the stability of duplex formation. Three of eight possible 3' single-nucleotide overhangs on a wobble terminal base pair have a significant stabilizing effect (CUG/G, CGA/U, and CGG/U) of 0.4 or more kcal/mol relative to 3' single-nucleotide overhangs on Watson-Crick terminal base pairs. Of 24 completed sequences, three 3' double-nucleotide overhangs on wobble terminal base pairs also have a stabilizing effect of 0.4 or more kcal/mol (CGGC/U, CUUA/G, and CUUC/G) relative to 3' single-nucleotide overhangs on Watson-Crick terminal base pairs. The thermodynamic parameters are also reported for 8 self-complimentary RNA duplexes in 1 M NaCl that contain 5' single-nucleotide overhangs on wobble terminal base pairs. Compared to 5' single-nucleotide overhangs on Watson-crick terminal base pairs, no significant stabilization is observed, nor a significant different in stabilizing effects.

Keywords: thermodynamics, wobble, overhangs

30. Differential Effects of HIV-1 NC on Strand-exchange Reactions Involving Mismatched DNA Duplexes

Besik Kankia (Department of Chemistry, The Ohio State University), Karin Musier-Forsyth

Abstract:
The HIV-1 nucleocapsid protein (NC) facilitates strand-replacement reactions during reverse transcription. The role of NC in these processes is linked to its nucleic acid chaperone activity, whereby nucleic acids are rearranged into their thermodynamically most stable structure. In the present work, we use an optical quadruplex displacement assay to study the role of NC in strand-replacement reactions. Short DNA duplexes containing two G-A mismatches at different positions were investigated: adjacent mismatches in the middle of the duplex (Reaction 1), single mismatches separated by 11 nucleotides (Reaction 2), and adjacent mismatches at the end of the duplex (Reaction 3). The reactions were studied as a function of temperature and strand concentration, which revealed that strand replacement proceeds through two different pathways: dissociative (monomolecular) and sequential displacement (bimolecular). The former requires complete dissociation of the reactant duplex followed by rapid formation of product duplex, and the latter pathway assumes that the chase strand initially base pairs to the reactant duplex at the most unstable part followed by a fast displacement process. As expected, the activation energy of the sequential displacement pathway is lower than that of the dissociative pathway for Reactions 1 and 2. Surprisingly, in the case of Reaction 3 the opposite is true. We hypothesize that this is due to the formation of a bimolecular intermediate, most likely a three-stranded DNA duplex stabilized by G-quartets. NC accelerates the strand-replacement kinetics by 1-2 orders of magnitude, and lowers the activation energy of the monomolecular pathway by 10-15 kcal/mol for all reactions. NC also lowers the activation energy of the sequential displacement pathway for Reaction 1 and 2 by 25 kcal/mol and 13 kcal/mol, respectively. In the presence of NC, the dissociative pathway completely dominates the kinetics of Reaction 3, which could be explained by more favorable formation of the bimolecular intermediate due to NC’s nucleic acid aggregating ability.

Keywords: DNA strand-exchange, HIV-1 nucleocapsid protein, Kinetics

31. Biochemical and thermodynamic characterization of guanosine substrate-binding of a group I Intron from bacteriophage Twort

Hajeong Kim (Department of Biochemistry, Purdue University), Josh Sokoloski (Department of Chemistry, Penn State University), Philip C. Bevilacqua (Department of Chemistry, Penn State University), Barbara L. Golden (Department of Biochemistry, Purdue University)

Abstract:
The group I intron from the bacteriophage Twort binds guanosine (G) very tightly, much tighter than the Anabaena and Tetrahymena group I introns. This is surprising because group I intron active sites are very highly conserved. To explore why this intron might bind G so tightly, we first examined the possibility that these differences are originated from a little sequence variation in G-binding site. In a Twort mutant ribozyme that has the sequences of the top layer of G-binding site of the Tetrahymena ribozyme, guanosine dissociation constant increases 4-fold. This is consistent with the finding that in the Tetrahymena ribozyme, there is less stacking between G-substrate and the active site residues than in other ribozymes. As stacking is one of the major forces to stabilize nucleic acid structure, this partly explains our inquiry. To further examine the origin of the differences in G binding, we characterized the thermodynamics of G binding by isothermal titration calorimetry and kinetic experiments. Surprisingly, the Twort ribozyme behaves differently than other group I introns. The Tetrahymena and Anabaena ribozymes bind G in an entropically favored process where î H = ~0. In contrast, G binding by the Twort intron is enthalpically favored. To explain these unexpected differences, we hypothesize that the unbound conformation of G-binding site of the Twort ribozyme is more native-like while in the other group I introns, the site is nearly collapsed. This may be due at least in part to the presence of P7.1-7.2 subdomain that may stabilize the conformation of the active site.
Neurospora mt tyrosyl-tRNA synthetase protein (CYT-18) recognizes and stabilizes the core structure of group I introns. Several lines of evidence suggest that CYT-18 stabilizes the conformation of the G-binding site of group I introns. To examine this possibility, we determined Mg2+ concentration dependence of G-binding constant (KMG). While KMG in the absence of CYT-18 was greatly increased with decreasing Mg2+concentration, in the CYT-18-assisted reaction, it was nearly constant. This suggests that CYT-18 stabilizes the three-dimensional structure of G-binding site at low Mg2+concentrations. The number of Mg2+ions required for catalysis was estimated by studying the Mg2+concentration dependence of kcat. The number of Mg2+ions binding in the absence of CYT-18 is much higher than that in the presence of CYT-18. This suggests that influence of structural Mg2+ions be at least lowered in presence of Cyt-18, but that the protein cannot compensate for catalytic Mg2+ ions. This system may allow us to probe the roles of the catalytic Mg2+ions.

Keywords: group I Intron, thermodynamics, guanosine binding

32. A comparison of two methods used to investigate peptide:RNA heteroconjugates by mass spectrometry

Kady L. Krivos (Chemistry, University of Cincinnati), Larry Sallans (Chemistry, University of Cincinnati), Patrick A. Limbach (Chemistry, University of Cincinnati)

Abstract:
Protein:RNA complexes participate in transcription, translation (ribosomes), chaperoning (signal recognition particles), and enzymatic cleavages (ribozyme and splicesome) to name a few. To date, the biochemical methods for obtaining sequence and structural information on these complexes have been arduous tasks often involving hazardous radioactive labeling and some the methods lack the ability to identify peptide or RNA modifications. Mass spectrometry has previously been developed as a method to investigate peptide:oligonucleotide complexes, however it has been shown to result in the preferential loss of the modifications. A recently developed method of fragmentation called electron capture dissociation (ECD) has been employed in recent years in mass spectrometry as a fragmentation technique of peptides because it can provide more extensive sequence coverage and improved identification of the location of labile post translational modifications when compared to CID. The focus of the present work is a comparison of the two fragmentation techniques, CID and ECD, to see if more sequence information can be elucidated for peptide:oligonucleotide heteroconjugates in mass spectrometry. In particular, we seek to learn if ECD will provide a more effective approach for identifying amino acids containing the site of RNA cross-linking. Synthetic heteroconjugates containing a 2-5 oligonucleotides covalently linked through amide chemistry to peptide sequences less than 15 amino acids in length have served as the model systems for this investigation. CID of these complexes has revealed the propensity for sequencing the RNA portion of the heteroconjugate and has not allowed for the identification of the point of crosslinking. Initial ECD studies have shown the fragmentation and sequencing of the peptide moiety to be favored. On going investigation of ECD as well as the heteroconjugate properties are being investigated to optimize this method.

Keywords: RNAProtein, mass spectrometry

33. Investigating interactions of the putative endonuclease Nob1 with 18S rRNA

Allison C. Lamanna (Chemistry Department, University of Michigan), Katrin Karbstein (Chemistry Department, University of Michigan)

Abstract:
Nob1 is an essential Saccharomyces cerevisiae protein involved in ribosome biogenesis. Specifically, cells depleted in Nob1 show a defect in processing of the 40S small ribosomal subunit rRNA: cleavage of 20S to form the mature 3’ end of 18S rRNA does not occur (1). Because Nob1 contains a predicted PIN domain that is required for this cleavage (2), it has been proposed that Nob1 is the endonuclease responsible for maturation of the 20S rRNA to 18S rRNA. Here, we show that Nob1 binds to rRNA fragments from the 3’ end of the 20S rRNA. Additionally, while Nob1 is a monomer in solution, it oligomerizes on the rRNA substrate. Homology modeling of the Nob1 PIN domain has suggested that Nob1 can form a tetramer through interactions in this domain (2). Further work will explore the ability of Nob1 to cleave 20S rRNA fragments and the stoichiometry of the Nob1-rRNA interaction.

References:
(1) Fatica, A., Oeffinger, M., Dlakic, M., and Tollervey, D. (2003) Mol. Cell. Biol. 23, 1798-1807.
(2) Fatica, A., Tollervey, D., and Dlakic, M. (2004) RNA 10, 1698-1701.

Keywords: endonuclease, ribosome biogensis, Saccharomyces cerevisiae

34. Single Molecule FRET to Explore Protein-RNA Interactions in the Central Domain of the 30S Ribosomal Subunit

Rajan Lamichhane (Department of Chemistry, Wayne State University, Detroit, MI 48202), Kris A. Baker (Department of Biological Sciences, Wayne State University, Detroit, MI 48202), Philip R. Cunningham (Department of Biological Sciences, Wayne State University, Detroit, MI 48202), David Rueda (Department of Chemistry, Wayne State University, Detroit, MI 48202)

Abstract:
The central domain of the 16S rRNA contains several protein binding sites. S15 is a central domain primary binding protein that has been shown to trigger a conformational change in the rRNA.1 This conformational change allows other central domain binding proteins to bind to the central domain causing a cascade of changes resulting in the functional structure of the central domain. Previous biochemical and structural studies have revealed two regions that are minimally needed for binding S15 in vitro. One of the regions is the junction of helices 20, 21, and 22 in 16S rRNA, which includes nucleotides 652-654 and 752-754 plus two or three base pairs in each helix to maintain the junction structure.

To identify functionally important sequence and structural elements within the junction loop, nucleotides 652-654 and 752-754 were subjected to saturation mutagenesis and functional mutants were selected and analyzed. To determine if S15 binding was affected by mutations in the junction loop, S15 was cloned and over-expressed with the junction mutations. S15 complemented mutations in the junction loop in each of the partially functional mutants. Nonfunctional mutants were not complemented by over-expression of S15.

Individual mutants were analyzed using single molecule Fluorescence Resonance Energy Transfer (smFRET) in vitro. Comparison of the structural dynamics of these mutants to that of the WT sequence in the presence and absence of S15 has revealed specific sequence and structural motifs in the junction loop that are important in ribosome function.

References:
1. Agalarov, S. C., Sridhar Prasad, G., Funke, P. M., Stout, C. D. & Williamson, J. R. (2000). Structure of the S15,S6,S18-rRNA complex: assembly of the 30S ribosome central domain. Science 288, 107-13.

Keywords: S15, Ribosome, smFRET

35. Role of modified nucleotides in helix 31 of Escherichia coli 16 S ribosomal RNA

Tek N. Lamichhane (Department of Chemistry/Biological Sciences, Wayne State University), Ashesh A. Saraiya (Department of Pharmaceutical Chemistry, University of California, San Francisco), Christine S. Chow (Department of Chemistry), Philip R. Cunningham (Department of Biological Sciences, Wayne State University)

Abstract:
The 970 loop of helix 31 is highly conserved within Bacteria, Archaea and Eukarya, but only A964, A969, and C970 are conserved among all three domains. The 970 loop also contains two of the eleven modified nucleosides in E. coli 16 S rRNA, m2G966 and m5C967. Biochemical and structural studies have placed this loop near the P-site and have shown that it is involved in the decoding process and in binding the antibiotic tetracycline. To identify the function of modified nucleotides in this loop, mutations were constructed at m2G966 and m5C967 and tested for ribosome function in vivo using the specialized genetic system developed in the Cunningham lab. Analyses of the mutants revealed that single mutations at positions 966 and 967 produce ribosomes that are approximately 20%-30% more active than wild-type ribosomes. Biochemical and modeling studies suggest that initiation factor 3 (IF3) interacts with the stacked residues 966:967:968 in H31. Over-expression of IF3 specifically restores wild-type levels of protein synthesis to the 966 and 967 hyperactive mutants. These data suggest that m2G966 and m5C967 may affect IF3 binding to the 30 S subunit. To determine the effect of these hyperactive mutations on translational fidelity, the mutant ribosomes were tested for mistranslation by starving wild-type and mutant plasmid containing cells for asparagine and measuring amino acid misincorporation in by isoelectric focusing. These data show that the m2G966U mutation produces ribosomes that are significantly more error prone than wild-type ribosomes. All 3 mutations at positions 966 and 967 were also tested for read-through of the UAG and UGA stop codons in vivo using luciferase premature termination constructs. All 966 mutants produced increased read-through of both codons but less effect on read-through was observed with the 967 mutants. From structural studies, it is reported that m2G966 and C1400 are involved in forming a binding cavity for P-site tRNA and the distance between these two nucleotides is decreased upon tRNA binding. To determine if a functional interaction exists between m2G966 and C1400, a series of double mutants at these positions were constructed and analyzed. No significant difference in function between the double and single mutants were found. This results suggest that a functional interaction between m2G966 and C1400 does not exist.

Keywords: Ribosomal RNA, Modifying enzymes, Aminoacid starvation

36. Comparison of the Catalytic Activity of Human and Yeast snRNAs

Caroline Lee (RNA Center, Case Western Reserve University)

Abstract:
We previously showed that protein-free fragments of human U6 and U2 snRNAs formed a basepaired complex that resembled the one forming in the activated spliceosomes, and further, provided evidence for splicing-like catalysis in this system. To extend the characterization of the catalytic activity of these two snRNAs, we have started a detailed comparison of the structural features and catalytic activity of U6 and U2 snRNAs derived from human and yeast. The RNAstructure algorithm indicated that while the global base-paired structure of the U6/U2 complex were similar in yeast and human, a number of structural elements, such as U6/U2 helix I and the U2 stemloop I showed a different basepairing pattern. In addition, the conserved ACAGAGA box of U6 was predicted to form a basepaired structure in the yeast U6/U2 complex, while in human this region remains unpaired. To determine the role of these structural differences, we compared the catalytic activity of human and wild type and mutant yeast U6/U2. While these mutations caused very minimal alteration of the primary sequence of yeast U6 and U2, they induced individual domains of the yeast U6/U2 complex to form basepairing interactions similar to those predicted in the human complex. While wild-type and mutant yeast U6/U2 did not differ in substrate binding, catalytic assays indicated that the wild type yeast U6/U2 and all except one of the mutant yeast U6/U2 complexes could not perform splicing-related catalysis in vitro. Intriguingly, the only yeast U6/U2 mutant that showed catalytic activity was the one that resembled the human U6/U2 complex most in terms of its secondary structure. This suggests that the differences in primary sequence of human and yeast U6 and U2 are not incompatible with catalytic activity, and that the secondary structure of the U6/U2 complex makes crucial contributions to the catalytic function of these RNAs.

Keywords: U6U2, Splicing, Catalysis

37. Pathogenic mechanism of a human mitochondrial tRNAPhe mutation associated with MERRF syndrome

Jiqiang Ling (Department of Microbiology and the Ohio State Biochemistry Program, the Ohio State University), Herve Roy, Mary Anne T. Rubio (Department of Microbiology, the Ohio State University), Daoming Qin, Juan D. Alfonzo, Kurt Fredrick (Department of Microbiology and the Ohio State Biochemistry Program, the Ohio State University), Michael Ibba (Department of Microbiology and the Ohio State Biochemistry Program, the Ohio State University)

Abstract:
Human mitochondrial tRNA (hmt-tRNA) mutations are associated with a variety of diseases including mitochondrial myopathies, diabetes, encephalopathies, and deafness. Since the current understanding of the precise molecular mechanisms of these mutations is limited, there is no efficient method to treat their associated mitochondrial diseases. Here, we utilize a variety of known mutations in hmt-tRNAPhe to investigate the mechanisms that lead to malfunctions. We tested the impact of hmt-tRNAPhe mutations on aminoacylation, structure, and translation elongation factor binding. The majority of the mutants were pleiotropic, exhibiting defects in aminoacylation, global structure and elongation factor binding. One notable exception was the G34A anticodon mutation of hmt-tRNAPhe (mitochondrial DNA mutation G611A), which is associated with myoclonic epilepsy with ragged red fibers (MERRF). In vitro the G34A mutation decreases aminoacylation activity by 100-fold, but does not affect global folding or recognition by elongation factor. Furthermore, G34A hmt-tRNAPhe does not undergo adenosine-to-inosine (A-to-I) editing ruling out mis-coding as a possible mechanism for mitochondrial malfunction. To improve the aminoacylation state of the mutant tRNA, we modified the tRNA binding domain of the nucleus-encoded human mitochondrial phenylalanyl-tRNA synthetase, which aminoacylates hmt-tRNAPhe with cognate phenylalanine. This variant enzyme displayed significantly improved aminoacylation efficiency for the G34A mutant, suggesting a general strategy to treat certain classes of mitochondrial diseases by modification of the corresponding nuclear gene.

References:
Ling,J., Roy,H., Qin,D., Rubio,M.A., Alfonzo,J., Fredrick,K., & Ibba,M. Pathogenic mechanism of a human mitochondrial tRNAPhe mutation associated with MERRF syndrome. Proc. Natl. Acad. Sci. USA. In press.

Keywords: tRNA, mitochondrial disease, gene therapy

38. Isolation of the tetrahymena telomeric protien complex

Benjamin R. Linger (Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati), Carolyn M. Price (Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati)

Abstract:
In order to maintain genome integrity, the telomeric DNA from cells with linear chromosomes is packaged into a protective nucleoprotein complex. In the absence of such a complex, the telomeres are recognized as DNA damage and subject to repair by non-homologous end joining (NHEJ). The resulting chromosome fusions lead to profound genome instability. Although telomeric nucleoprotein complexes are known to contain a variety of unique telomere proteins plus general DNA repair factors, the full protein complement of a telomere has not been determined for any organism. Our goal is to isolate a native telosome with all its component proteins. We are using the ciliate Tetrahymena thermophila due to the abundance of genetic tools available and its atypical biology that generates thousands of telomeres per cell. To isolate a native telosome, we have engineered the rDNA chromosome so that the entire telosome can be released by restriction digestion. This chromosome is an excellent source for telosome isolation because of its high copy number of ~10,000 molecules per cell. We have shown that the telosome can be released from the engineered rDNA by digestion with PvuII. We have also verified that the telomere protein POT1a associates with the releasable telomeres. To promote affinity purification of released telomeres, we subsequently added a TAP-tag to the N-terminus of the POT1a gene in the cell line with the engineered rDNA chromosome. Initial immunoprecipitation of soluble TAP-POT1a and subsequent mass spectrometry has identified two potential POT1a binding partners for which we are now pursuing functional analysis. The homology observed between telomere proteins identified to date suggests that characterization of the Tetrahymena telosome components will facilitate identification of key components of human telomeres and provide insight into how these components maintain functional telomeres that are essential for genome stability.

Keywords: telomerase, telomere

39. Hsp90 control of mRNA decay through stabilization of the PMR1 mRNA endonuclease

Yong Peng (Department of Molecular and Cellular Biochemistry; The Ohio State University; Columbia University), Xiaoqiang Liu (Department of Molecular and Cellular Biochemistry; The Ohio State University), Daniel R. Schoenberg (Department of Molecular and Cellular Biochemistry; The Ohio State University)

Abstract:
The PMR1 mRNA endonuclease forms a selective complex with its translating
substrate mRNAs where it is activated to initiate mRNA decay. Previous work showed
tyrosine phosphorylation is required for PMR1 targeting to this polysome-bound
complex, and identified c-Src as the responsible kinase. c-Src phosphorylation occurs in
a distinct complex, and the current study identifies Hsp90 as a critical member of this.
Hsp90 binds to the central domain of PMR1 and its binding is inhibited by geldanamycin.
Geldanamycin stabilizes substrate mRNA to PMR1-mediated decay and causes the
mRNA endonuclease to rapidly disappear. We show that PMR1 is inherently unstable,
that it is degraded by the 26S proteasome, and that geldanamycin activates a pre-existing
decay process. Lastly, we show that PMR1 is ubiquitinated, its ubiquitination is
stimulated by geldanamycin, and this activates its degradation. c-Src is regulated
similarly by Hsp90, and results of this study identify a new process controlling mRNA
decay through changes in effector protein stability.

Keywords: PMR1, endonuclease-mediated mRNA decay, Hsp90

40. Investigation of the structural roles of nucleic acid modification in the bacterial decoding region of 16S rRNA

Santosh K. Mahto (Department of Chemistry, Wayne State University, Detroit , MI 48202), Christine S. Chow (Department of Chemistry, Wayne State University, Detroit , MI 48202)

Abstract:
The translation of genetic information into proteins is a complex process performed by the macromolecule called the ribosome and is an essential process in all cells. The decoding region (helix 44) of 16S ribosomal RNA (rRNA) of the small subunit is a very important functional region; it is involved in binding to mRNA, tRNAs, and the large subunit rRNA. The decoding region is involved in translational accuracy, and is also responsible for translocation. In addition, some of the known antibiotics bind to this region. There are three modified nucleosides in the decoding site of E. coli 16S rRNA, namely 5-methylcytidine (m5C), 3-methyluridine (m3U), and N4, 2\'-O-dimethylcytidine (m4Cm). The functional significance of those modified nucleosides is still not completely understood. Out of the three modified nucleosides, m3U and m5C are commercially available. In contrast, m4Cm, one of the modified nucleosides in which both the base and sugar are methylated is not commercially available. In order to carry out detailed studies on the roles of m4Cm and additional analogues, N4-methylcytidine (m4C) and 2\'-O-methylcytidine (Cm), they were chemically synthesized. The phosphoramidite of m4Cm and its analogs m4C and Cm were prepared and a series of decoding region RNA models were constructed. UV melting was then carried out to determine the stabilizing effects of the methylated nucleosides within the context of the decoding region of 16S rRNA.

Keywords: Modified nucleosides, Decoding region, Ribosomal RNA

41. Characterization of the products of 5-fluorouridine after the action of E. coli pseudouridine synthase TruB

Edward J. Miracco (University of Louisville), Shi Bai (University of Delaware), Eugene G. Mueller (University of Louisville)

Abstract:
Pseudouridine synthases (&psi S) are the enzymes responsible for the conversion of U to &psi, with most &psi Ss specific for particular uridine residues. The most highly conserved &psi is located in the T &psi C loop of tRNA, and it is generated by TruB and its orthologs. Unlike many &psi Ss that are potently inhibited by RNA containing 5-fluorouridine ([f5U]RNA), TruB handles [f5U]RNA as a substrate, turning f5U into two hydrated products in a time frame similar to the natural conversion of U to &psi . The two products of f5U have been isolated after digestion with nuclease S1 and alkaline phosphatase and characterized by mass spectrometry and a set of 1D and 2D NMR experiments. The products are both dinucleotides, and both are rearranged isomers of f5U that have been hydrated. In agreement with the cocrystal structure of TruB and [f5U]RNA, the more abundant product is almost certainly (5S,6R)-5-fluoro-6-hydroxy-pseudouridine.

References:
Hoang, C., and Ferre-D’Amare, A. R. (2001) Cocrystal Structure of a tRNA Psi55
Pseudouridine Synthase Nucleotide Flipping by an RNA-Modifying Enzyme.,
Cell 107, 929-939.

Spedaliere, C. J., and Mueller, E. G. (2004) Not all pseudouridine synthases are
potently inhibited by RNA containing 5-fluorouridine, RNA 10, 192-199.

Spedaliere, C. J., Ginter, J. M., Johnston, M. V., and Mueller, E. G. (2004) The
Pseudouridine Synthases: Revisiting a Mechanism That Seemed Settled, JACS 126, 12758-12759.

Keywords: pseudouridine, fluorouridine, NMR

42. Probing the Role of Functional Domains of HIV-1 Nucleocapsid Protein in its Nucleic Acid Chaperone Function

Mithun Mitra (Chemistry, The Ohio State University), Daniel G Mullen (Chemistry, University of Minnesota), George Barany (Chemistry, University of Minnesota), Karin Musier-Forsyth (Chemistry, The Ohio State University)

Abstract:
The nucleocapsid protein from human immunodeficiency virus type-1 (HIV-1) functions as a nucleic acid chaperone protein by facilitating nucleic acid conformational changes to form the most thermodynamically stable arrangement. The nucleic acid chaperone activity of NC depends on its nucleic acid aggregating ability and weak duplex destabilizing activity. During the minus-strand transfer step of reverse transcription, NC facilitates the annealing of highly structured transactivation response region (TAR) RNA to the complementary TAR DNA. In this work, the role of different functional domains of NC in facilitating 59-nt TAR RNA/DNA annealing was probed by employing chemically synthesized peptides derived from full-length (55 amino acids) HIV-1 NC: NC(1-14), NC(15-35), NC(36-55), NC(1-35) and NC(29-55). All of these peptides, except NC(1-35), displayed significantly reduced annealing kinetics even when present at ~50-fold higher concentration than WT. The truncated NC constructs generally bind more weakly to single-stranded oligonucleotides, consistent with the loss of either electrostatic or hydrophobic contacts. In addition, the binding of the NC constructs was found to be slightly modulated by zinc-coordination to the CCHC motifs. Interestingly, NC(1-35), displayed annealing kinetics equal to that of full-length NC. NC(1-35) also displayed nucleic acid binding and aggregation activity that was indistinguishable from that of WT NC. Thus, we conclude that the N-terminal zinc finger flanked by the N-terminus and linker domains represents the minimal sequence necessary and sufficient for chaperone function. In contrast, a 14-residue peptide derived from the highly basic N-terminal domain of NC, which is known to be the major contributor to the nucleic acid aggregating activity of NC, was unable to effectively aggregate nucleic acids. Moreover, addition of NC(1-14) to NC(15-35) in trans does not stimulate aggregation or annealing activity of either construct. Therefore, covalent continuity of the N-terminal 35 amino acids of NC is critical for chaperone function.

Keywords: HIV-1, Nucleocapsid, Chaperone

43. Effect of tetraloop stability on the affinity of tRNA for T box antiterminator

Abigael M. Muchenditsi (Department of Chemistry and Biochemistry, Ohio University), Dr. Jennifer V. Hines (Department of Chemistry and Biochemistry, Ohio University)

Abstract:
The T box transcription antitermination mechanism involves a unique RNA/RNA interaction between uncharged cognate tRNA and the 5′ mRNA leader region of T box genes. One of the interactions is between the anticodon loop of the tRNA and a specifier sequence in the mRNA. The second interaction is between the acceptor end of the tRNA and the four nucleotides of a highly conserved mRNA structural element, known as the antiterminator. The formation of this second interaction prevents the formation of an alternative terminator structural element , thus leading to transcription antitermination and full synthesis of the gene. In a model antiterminator RNA a non-conserved loop in the antiterminator was replaced by the highly stable UUCG tetraloop. Similarly, a microhelix model tRNA was closed by the UUCG tetraloop. Binding and structural studies of the resulting antiterminator-microhelix complex indicated that the helices closed by the UUCG tetraloop are perturbed in, different manners, from standard A-form geometry. In order to determine if this deformation is functionally relevant and whether tRNA affinities are affected by the loop stabillity, a series of alternative loops were investigated in fluorescence-based tRNA binding assays.

Keywords: Transccription antitermination, fluorescence, tetraloop

44. Common SNP in pre-miR146a interferes with miR processing and predisposes to papillary thyroid carcinoma

Elizabeth L. Murray (Molecular and Cellular Biochemistry, The Ohio State University), Krystian Jazdzewski (Human Cancer Genetics Program, Comprehensive Cancer Center, The Ohio State University), Daniel R. Schoenberg (Molecular and Cellular Biochemistry, The Ohio State University), Albert de la Chapelle (Human Cancer Genetics Program, Comprehensive Cancer Center, The Ohio State University)

Abstract:
PTC is the most common malignancy in thyroid tissue. A strong inherited genetic predisposition is suggested by case-control studies, however, no predisposing genetic factors have been convincingly identified. Several microRNAs are up-regulated in PTC tumors, the most striking being a 19-fold increase in the quantity of miR146b. Human miR146 occurs in two distinct forms; miR146a and miR146b, whose mature forms differ by only 2 nucleotides. Therefore, both miRs share many predicted target genes, while each also has its own unique predicted targets.

To elucidate the role of miR146 in PTC we sequenced pri-pre-miR146a and pri-pre-miR146b from genomic DNA of patient samples that had been analyzed for global miR expression (He at al. 2005). No previously undescribed sequence changes were detected. However, pre-miR146a contained a common G/C polymorphism in the passenger strand that causes mispairing within the predicted hairpin structure. Sequencing of genomic DNA from PTC patients and controls showed differences in the distribution of genotypes between cancer patients and controls, with G/C heterozygosity associated with an increased risk of acquiring PTC in comparison with either homozygosity. Strikingly somatic mutations leading to heterozygosity are found in tumor tissue, suggesting that the germline genotype at the SNP affects the predisposition to PTC, and second, that the genotype at the SNP is frequently mutated in PTC tumors. To determine the molecular basis for this we examined the impact of the G/C polymorphism on miR146a expression. Pre-miR146a and the mature miR accumulate in cells expressing the G-containing allele but not in cells expressing the C-containing allele. There is no difference in protein binding to either pre-miR when assayed by UV crosslinking of complexes formed with HeLa cell nuclear extract; however, RNA EMSA showed that higher order complexes assemble on on pre-miR146a-G but not pre-miR146a-C, indicating that the latter is inefficiently processed to the mature miR. Thus quantitative changes in miR146a resulting from G/C heterozygosity are a predisposing factor in the development and/or growth of PTC.

Kaarle Franssila, Helsinki University Central Hospital, Helsinki, Finland and Barbara Jarzab, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice, Poland provided tissue samples for genetic analyses.

References:
He et al. 2005. The role of microRNA genes in papillary thryroid carcinoma. PNAS 102(52)19075-19080.

Taganov et al. 2006. NF-kappaB-dependent induction of microRNA miR-146, an inhibitor targeted to signaling proteins of innate immune responses. PNAS 103(33)12481-12486.

Keywords: microRNA, papillary thryoid carcinoma

45. Cytoplasmic capping of RNA decay intermediates

Yuichi Otsuka (Molecular and Cellular Biochemistry, The Ohio State University), Daniel R. Schoenberg (Molecular and Cellular Biochemistry, The Ohio State University)

Abstract:
β-thalassemia is an inherited disorder of hemoglobin production that results from the presence of a premature termination codon in the body of β-globin mRNA. In murine erythroleukemia (MEL) cells, PTC-containing β-globin mRNA is degraded by endonuclease cleavage and results in the accumulation of metastable mRNA decay intermediates. An earlier report suggested that these intermediates possess a cap-like modification at 5'end [Lim, S.K., and Maquat, L.E. 1992. EMBO J. 11:3271-3278]. The first part of this study examined the cap status of these decay intermediates by their recovery with an immobilized monoclonal antibody to the trimethylcap or a cytoplasmic cap binding protein eIF4E. Both full-length mRNA and the smaller decay products were recovered, supporting the presence of a true cap. This was confirmed by cap analog competition for binding to eIF4E, susceptibility to hydrolysis by tobacco acid pyrophosphatase, and removal of the cap with Dcp2. Mammalian capping enzyme (MCE) is generally thought to be restricted to the nucleus. Using a sensitive guanylylation assay, a cytoplasmic MCE-containing complex was identified that sediments at 140-200 kDa on glycerol gradients. Capping enzyme transfers a GMP to the 5'-diphosphate end of RNA via a covalent enzyme-GMP intermediate to generate the cap structure. However, the endonuclease that cleaves β-globin mRNA generates cleavage products with a '-monophosphate end. We present evidence for a heretofore unseen 5'-monophosphate kinase recovered with epitope-tagged capping enzyme from mammalian cells that facilitates addition of GMP onto RNA with a 5' monophosphate but not onto RNA lacking 5' phosphate. These results identify a potential new role for capping enzyme in modifying endonuclease-generated decay products, perhaps for subsequent decay.

Keywords: NMD, decay intermediates, capping enzyme

46. Deciphering the function of G-patch proteins in Saccharomyces cerervisiae

Sudakshina Paul (Biology and University of Kentucky), Li Zhang (Biology and University of Kentucky), Brian Rymond (Biology and University of Kentucky)

Abstract:
The G patch is a conserved motif composed of about 40 amino acids that is present in a number of RNA binding proteins. I identified five G annotated patch proteins in Saccharomyces cerevisiae, namely, Spp2p, Spp382p, Sqs1p, Pxr1p and the uncharacterized ORF YLR271w. Of these, Spp2p and Spp382p interact with the splicing-associated DEAD/H box proteins Prp2p and Prp43p respectively. The published data are consistent with Spp2p and Spp282p acting as recruitment factors or activators of Prp2p and Prp43p, respectively. Pxr1p has been implicated in rRNA and snoRNA processing. The function(s) of the remaining two G patch proteins Sqs1p and Ylr271wp is unknown although neither is essential for cell viability. To begin my investigation of function, I compared the growth and splicing in yeast that overexpress each of the G-patch proteins. Our data reveal that overexpression of three (SPP382,SQS1 and PXR1) of the five G-patch proteins impair yeast growth at 30C. Overexpression of (at least) (SPP382,SQS1 and YLR271w impairs mRNA processing of intron-bearing transcripts, possibly through inhibition of one of the DExD/H-box proteins. No splicing defect was found in yeast deleted for either SQS1 or YLR271w.

Since Prp43p (the Spp382p–associated protein) is required for ribosome biogenesis as well as splicing, I also investigated the impact of overexpression of each factor on the Prp43-dependent cleavage of the 35S rRNA. While no clear inhibition was observed, we do see suppression of a spp382 (splicing factor) mutant when the ribosome biogenesis factor, Pxr1p is overexpressed. These and related observations are used to support a model for selective partitioning of Prp43p function in the nucleolus and nucleoplasm through association with alternative G-patch targeting factors.

Keywords: splicing, G-patch, Saccharomyces cerevisiae

47. Characterization of 16S rRNA mutations that decrease the fidelity of translation initiation

Daoming Qin (The Ohio State Biochemistry Program, The Ohio State University), Nimo M. Abdi (Department of Microbiology, The Ohio State University), Kurt Fredrick (The Ohio State Biochemistry Program, Department of Microbiology, The Ohio State University)

Abstract:
In bacteria, initiation of translation is kinetically controlled by factors IF1, IF2, and IF3, which work in conjunction with the 30S subunit to ensure accurate selection of the initiator tRNA (fMet-tRNAfMet) and the start codon. Here, we show that mutations G1338A and A790G of 16S rRNA decrease initiation fidelity in vivo and do so in distinct ways. Mutation G1338A increases the affinity of tRNAfMet for the 30S subunit, suggesting that G1338 normally forms a suboptimal Type II interaction with fMet-tRNAfMet. By stabilizing fMet-tRNAfMet in the pre-initiation complex, G1338A may partially compensate for mismatches in the codon-anticodon helix and thereby increase spurious initiation. Unlike G1338A, A790G decreases the affinity of IF3 for the 30S subunit. Thus A790G may indirectly stabilize fMet-tRNAfMet in the pre-initiation complex and/or promote premature docking of the 50S subunit, resulting in increased levels of spurious initiation.

Keywords: ribosome, 16S rRNA, P site

48. The Nucleocapsid Protein of Feline Immunodeficiency Virus is an Efficient Nucleic Acid Chaperone

Dominic Francis Qualley (Department of Chemistry and Biochemistry and the Center for Retrovirus Research), Karin Musier-Forsyth (Department of Chemistry and Biochemistry and the Center for Retrovirus Research)

Abstract:
Nucleic acid chaperone proteins facilitate DNA and RNA rearrangements resulting in a thermodynamically more stable conformation. In retroviruses, the small, highly basic nucleocapsid (NC) protein serves as a chaperone and facilitates many nucleic acid restructuring events in the retroviral life cycle. Feline immunodeficiency virus (FIV) is a retrovirus that naturally occurs in the domestic cat and produces symptoms similar to those found in HIV-infected humans. FIV is very similar to HIV in structure and pathology and is a useful animal model for the study of certain aspects of pathogenesis and for the evaluation of antiviral strategies. Furthermore, the use of FIV as a tool for gene therapy makes it a particularly attractive model system in which to test such approaches. Compared to HIV, little is known about the molecular determinants of FIV NC chaperone activity. In this work, we carry out biochemical and biophysical studies to investigate FIV NC’s chaperone function. Sedimentation assays were used to characterize NC-facilitated nucleic acid aggregation, gel mobility-shift assays measured NC-guided annealing of DNA/RNA, and fluorescence anisotropy experiments provided apparent Kd values for FIV NC binding to DNA and RNA constructs. Taken together, the studies carried out to date provide evidence that FIV NC protein shows chaperone activity similar to that of HIV NC. Future experiments are aimed at mutational analysis of FIV NC in vitro and in cell culture.

Keywords: retrovirus, nucleocapsid

49. Expression and preliminary studies of YNL022p, a putative rRNA methyltransferase.

Kent L. Redman (Indiana University School of Medicine-Fort Wayne), Jennifer L. Craft (Department of Biology, Ball State University)

Abstract:
Posttranscriptional modifications in Saccharomyces cerevisiae produce 5-methylcytosine (m5C) in several tRNAs and in 25S rRNA. Trm4p methylates tRNA, so either or both of the putative RNA m5C methyltransferases encoded by the yeast genome (Nop2p and YNL022p) could be responsible for rRNA methylation. To initiate studies of YNL022p, the corresponding open reading frame was amplified and inserted into the pET28b vector so that the expressed protein includes a His6 tag. Only insoluble protein was formed after induction of BL21(DE3) cells at 37 oC. Protein solubility was maximal, yet poor when YNL022c was expressed at 30 oC. An adequate level of YNL022c expression for analysis was achieved by coexpression of the His6 tagged construct with bacterial chaperone proteins at 30 oC.
Assays measuring the incorporation of radiolabel from 3H-[methyl]-AdoMet into total RNA revealed greater incorporation of isotope into RNA with extracts from YNL022p expressing cells than with control extracts. However, only marginally more radiolabel was introduced into methyl-deficient RNA isolated from yeast lacking YNL022c than was incorporated into methyl-replete RNA. To characterize methylated RNAs more specifically, assay mixtures were analyzed by SDS-PAGE. Control and YNL022p extracts methylated tRNA-sized molecules, but only the YNL022p extract generated a large methylated product. Further specificity was indicated by the production of the large product only with methyl-deficient RNA. Since large RNAs are not well resolved by SDS-PAGE, methylated RNAs were separated in denaturing agarose gels. Like the previous experiments, only YNL022p preparations were found to cause the methylation of a large RNA and the radiolabeled RNA comigrated with the 25S rRNA marker. Therefore, YNL022c appears to be a rRNA m5C methyltransferase. When partially purified rRNA is used as the substrate, a small RNA molecule is highly methylated by the YNL022p extract indicating the possibility of an additional substrate for the enzyme. Further analysis of YNL022p is in progress.

Keywords: methylation, 5-methylcytosine, RNA

50. Alternative mechanisms of determining the specificity of the genetic code in the cytoplasm and mitochondria.

Noah Reynolds (Department of Microbiology, The Ohio State University), Herve Roy (Department of Microbiology, The Ohio State University), Jiqiang Ling (Ohio State Biochemistry Program, The Ohio State University), Michael Ibba (Department of Microbiology and Ohio State Biochemistry Program, The Ohio State University)

Abstract:
The fidelity of translation is mainly determined by two events: synthesis of cognate amino acid:tRNA pairs by aminoacyl-tRNA synthetases (aaRSs) and accurate selection of aminoacyl-tRNAs (aa-tRNAs) by the ribosome. AaRSs define the genetic code by pairing tRNAs with the corresponding amino acids. To ensure faithful aa-tRNA synthesis, many aaRSs employ an additional editing activity that hydrolyzes incorrectly activated non-cognate amino acids. Eukaryotic organisms contain two distinct phenylalanyl-tRNA synthetases (PheRS), a cytoplasmic (ctPheRS) and a mitochondrial form (mtPheRS). CtPheRS is similar to the E. coli enzyme in that it consists of a heterotetramer made up of two alpha-beta heterodimers in which the alpha-subunits contain the active site and the beta-subunits contain the editing site. MtPheRS, as well as the chloroplast form, is an alpha-subunit monomer and lacks a recognizable editing domain, consistent with the absence of editing in some other mt aaRS. It has been shown that mtPheRS does not have the ability to edit mischarged tRNAPhe, nor do mitochondria contain any trans editing activity able to compensate for this deficiency. The two forms of PheRS differ both in their ability to edit Tyr-tRNAPhe and in the specificity of their amino acid binding sites. Amino acid activation kinetics show that mtPheRS is 180-fold more selective for Phe over Tyr than is ctPheRS. Even though mtPheRS does not edit, it will produce only one Tyr-tRNAPhe per ~8000 Phe-tRNAPhe synthesized when Phe and Tyr are present at comparable concentrations, as is the case under standard growth conditions. This error rate is close to the level of 10-4 usually cited for protein synthesis, and begs the question as to whether editing functions outside translation since PheRS can achieve adequate substrate specificity in its absence in the mitochondria.

Keywords: Aminoacyl-tRNA synthetase, Genetic code, Translation

51. A New Role for Cisplatin: Probing the Ribosome Structure

Keshab Rijal (Department of Chemistry, Wayne State University), Christine S. Chow (Department of Chemistry, Wayne State University)

Abstract:
The ribosome is a potential target for several classes of antibiotics because of its accessibility and structural diversity. Different classes of antibiotics bind to different sites within the ribosome to inhibit the ribosomal assembly and translation process. Bacteria have become resistant to most of the known antibiotics; therefore, it is important to design and develop new therapeutic agents. The first step towards the development of drugs is to find ideal targets sites that are not only functionally important, but also accessible to various compounds. Knowledge of RNA structure and its relationship to function is also essential for interpretation of the biological mechanisms of RNA action. Most of the available chemical probes are limited to in vitro studies and detection of their reactive sites is challenging. We have utilized a new tool to probe RNA structure. Cisplatin is a well known anticancer drug that forms different adducts with DNA, RNA and proteins. Here, we have used cisplatin to probe accessible guanine residues in RNA model systems, 16S rRNA and the ribosome. This method has several advantages over other chemical probes: the adducts are easily detected, and the probe can be used in vivo.

Keywords: Probing, Cisplatin, Ribosome

52. Functional features of nuclear pre-mRNA introns containing miRNA coding regions

Siddharth Patil (BGES, Cleveland State University), Jinani Slaibi (BGES, Cleveland State University), Jason Rozick (BGES, Cleveland State University), Tiffany Kaul, Neha Aggarwal, Kavleen Sikand, Girish Shukla (BGES, Cleveland State University)

Abstract:
Numerous cell processes including transcription, nuclear pre-mRNA splicing, nonsense mediated decay, 5’ and 3’ end modification of mRNA and translation are influenced by a variety of cis-acting regulatory sequences found in human genome. It is expected that human genome encodes numerous other elements and motifs which may support optimal gene expression. Therefore the identification of such cis-regulatory elements and their functional significance is crucial to understand their role in human gene expression. A number of recently discovered noncoding RNAs known as micro (mi) RNAs are encoded within the coding (exon) and noncoding (intron) regions of a variety of functionally important human and other mammalian genes. We are interested in decoding the roles of these cis-acting sequences which comprise miRNAs ‘genomic neighborhoods’ in the overall gene expression cascade. Using bioinformatics approach we are attempting to identify, catalog and evaluate the function of these sequence elements that are found within the neighboring exons and introns of 85 human and 7 mouse miRNA host introns. Using a genome wide comparative genomics approach, currently we are developing a dataset that would eventually contain the comprehensive information with respect to human and other mammalian intronic miRNAs. The dataset is comprised of the information including the size of introns, size of exons, intronic splicing silencers, exonic splicing enhancers, consensus 5’ and 3’ splice sites, and various RNA binding protein region information. The identification and functional evaluation of these regulatory motifs and elements would help us to understand their role in coordinated events of nuclear pre-mRNA splicing and miRNA processing. Additionally, experimental analysis of functionally important regulatory noncoding miRNA molecules may provide better understanding of gene regulation, drug target discovery, and clinical prognosis for important disease states including cancer.

Keywords: miRNAs, bioinformatics

53. Probing RNA binding by the nucleoprotein from ebolavirus

Samuel S. Rund (Chemistry & Biochemistry, DePauw University), Jonathan M. Howard (Chemistry & Biochemistry, DePauw University), Sharon M. Crary (Chemistry & Biochemistry, DePauw University)

Abstract:
Ebolavirus is an enveloped, nonsegmented, negative-sense RNA virus. As such, its RNA genome must be tightly wrapped in nucleocapsid proteins to be replication competent; it is this ribonucleoprotein complex that is the template for the viral polymerase. The most abundant protein in the nucleocapsid, the nucleoprotein (NP), coats the entire ~19 kilobase genomic and antigenomic RNAs throughout the viral replication cycle. The mechanism by which NP encapsidates the RNA is unknown, however, it is likely that encapsidation is nucleated through interactions with specific non-coding regions at the 5\' end of the viral RNA. We have studied the interactions of the 5\' terminus of the antigenomic viral RNA with bacterially expressed soluble NP in vitro using electrophoretic mobility shift assays. Results from these binding experiments will be presented.

Keywords: Ribonucleoprotein, virus

54. Post-transcriptional modifications in bacterial rRNA and heat shock

Susan Russell (University of Cincinnati), Dr. Patrick A. Limbach (University of Cincinnati)

Abstract:
Post-transcriptional modifications found in ribosomal RNA are thought to be important for both the structure and function of the ribosome. The modified nucleosides of rRNA tend to cluster in the functionally important region of the ribosome and modifications occur in conserved regions. Few rRNA modifying enzymes are known and their regulation is not well understood. At least one modifying enzyme, FtsJ, a specific methyltransferase which creates the 2\'-O-methyluridine at U2552 in 23S RNA, has been identified as a heat shock protein.
The heat shock response is a mechanism which allows a cell to cope with a sudden increase in temperature or other environmental stress. The initiation of the heat shock response is transcriptionally regulated by an alternative sigma factor that directs RNA polymerase to different promoter sites which induces a rapid and transient expression of many genes that code for \

Keywords: RNA modification

55. Effects of antibiotics on spontaneous reverse translocation

Shinichiro Shoji (Department of Microbiology, The Ohio State University), Maria A. Borovinskaya (Physical Biosciences Division, Lawrence Berkeley National Laboratory), Jamie H. Daudna Cate (Physical Biosciences Division, Lawrence Berkeley National Laboratory), Kurt Fredrick (Department of Microbiology, Ohio State Biochemistry Program, The Ohio State University)

Abstract:
A number of antibiotics are known to inhibit EF-G-dependent translocation by interacting with the ribosome. Inhibition may be due to the ability of the antibiotic to stabilize the pretranslocation (PRE) complex, destabilize the transition state of translocation, or confer both of these abilities. We have measured the effect of several translocation inhibitors on spontaneous reverse translocation to help distinguish which potential mechanism is responsible for inhibiting EF-G-dependent (forward) translocation. Tetracycline, spectinomycin, and hygromycin B inhibit spontaneous reverse translocation, suggesting that these antibiotics destabilize the transition state of tRNA-mRNA movement. Streptomycin, aminoglycosides of the neomycin group (neomycin, paromomycin, gentamicin), and viomycin do not inhibit spontaneous reverse translocation. Interestingly, hygromycin B and the latter antibiotics cause conversion of ribosomes from the POST to PRE state in the presence of EF-G and GTP, corroborating earlier evidence that they stabilize the PRE state. On the basis of the newly solved structures of the ribosome complexed with antibiotics, we propose the molecular mechanisms of inhibition of translocation by these antibiotics.

Keywords: ribosome, antibiotics, reverse translocation

56. Activity of miRNAs in prostate carcinoma cells

Kavleen Sikand (BGES, Cleveland State University), Jinani Slaibi (BGES, Cleveland State University), Neha Aggarwal (BGES, Cleveland State University), Girish Shukla (BGES, Cleveland State University)

Abstract:
The discovery of noncoding microRNAs (miRNAs) has opened up new opportunities to study RNA mediated processes to understand the detailed molecular biology of cancer. Aberrant expression of miRNAs has been correlated in various leukemias and cancers including lung, thyroid and colorectal cancer. In addition, microarray based expression analyses revealed the aberrant expression of a number of miRNAs in solid prostate tumors. Unfortunately, microarray based expression analyses are not adequate to demonstrate molecular mechanisms that are mediated by miRNAs in cancers. Function of miRNAs and their activities in prostate cancer is still an unexplored research area. We are investigating the expression and role of miRNA clusters in various cell culture models of prostate carcinoma. These clusters are aberrantly expressed in prostate carcinoma cells. We are investigating the overexpression of two miRNAs from an oncogene miRNA cluster 17-92 on chromosome 13. We also found that at least one miRNA, a member of 106-92 cluster on X-chromosome, is highly upregulated in response to hormone in prostate carcinoma cells LNCaP. Surprisingly, not all miRNAs that are encoded in a cluster are expressed suggesting the possibility of additional post-transcriptional mechanisms in miRNA cluster expression. The long term goal of this work is to investigate if a link exists between the expression patterns of miRNAs and the transition of prostate cancer to fatal androgen independent stage.

Keywords: miRNA, prostate cancer

57. Functional features of nuclear pre-mRNA introns containing miRNA coding regions

Siddharth Patil (BGES, Cleveland State University), Jinani Slaibi (BGES, Cleveland State University), Jason Rozick, Tiffany Kaul, Neha Aggarwal, Kavleen Sikand, Girish Shukla (BGES, Cleveland State University)

Abstract:
Numerous cell processes including transcription, nuclear pre-mRNA splicing, nonsense mediated decay, 5’ and 3’ end modification of mRNA and translation are influenced by a variety of cis-acting regulatory sequences found in human genome. It is expected that human genome encodes numerous other elements and motifs which may support optimal gene expression. Therefore the identification of such cis-regulatory elements and their functional significance is crucial to understand their role in human gene expression. A number of recently discovered noncoding RNAs known as micro (mi) RNAs are encoded within the coding (exon) and noncoding (intron) regions of a variety of functionally important human and other mammalian genes. We are interested in decoding the roles of these cis-acting sequences which comprise miRNAs ‘genomic neighborhoods’ in the overall gene expression cascade. Using bioinformatics approach we are attempting to identify, catalog and evaluate the function of these sequence elements that are found within the neighboring exons and introns of 85 human and 7 mouse miRNA host introns. Using a genome wide comparative genomics approach, currently we are developing a dataset that would eventually contain the comprehensive information with respect to human and other mammalian intronic miRNAs. The dataset is comprised of the information including the size of introns, size of exons, intronic splicing silencers, exonic splicing enhancers, consensus 5’ and 3’ splice sites, and various RNA binding protein region information. The identification and functional evaluation of these regulatory motifs and elements would help us to understand their role in coordinated events of nuclear pre-mRNA splicing and miRNA processing. Additionally, experimental analysis of functionally important regulatory noncoding miRNA molecules may provide better understanding of gene regulation, drug target discovery, and clinical prognosis for important disease states including cancer.

Keywords: miRNAs, bioinformatics

58. Molecular basis for thiamin pyrophosphate recognition and gene regulation by the THI-box riboswitch

Angela M. Smith (Department of Microbiology, The Ohio State University), Nancy Ontiveros-Palacios, Mario Soberon (Instituto de Biotecnologίa, Universidad Nacional Autόnoma de Mexico), Frank J. Grundy, Tina M. Henkin (Department of Microbiology, The Ohio State University), Juan Miranda-Rios (Instituto de Biotecnologίa, Universidad Nacional Autόnoma de Mexico)

Abstract:
Riboswitches are RNA elements that modulate expression of genes upon direct sensing of specific regulatory signals. These elements respond to a wide range of environmental signals such as temperature change, small molecules, and non-coding RNAs. The THI-box riboswitch regulates expression of genes involved in thiamin biosynthesis in response to thiamin pyrophosphate (TPP). In E. coli the thiM gene is regulated at the level of translation initiation by a THI-box riboswitch located in the 5’ leader of the mRNA. In the TPP-bound form, the RNA folds into a structure in which the Shine-Dalgarno (SD) sequence is sequestered through base pairing with an anti-SD (ASD) sequence. When TPP levels are low, the ASD sequence forms an alternate pairing with an upstream sequence, the anti-anti-SD (AASD), leaving the SD region accessible for ribosome binding. An in vivo expression analysis using translational fusions to a lacZ reporter was used to investigate the effects of TPP binding on gene expression using the wild-type THI-box RNA and mutant constructs containing nucleotide substitutions in the TPP-binding domain. Mutation of residues predicted to be involved in TPP binding resulted in a loss of TPP-dependent repression with two distinct phenotypes observed. Class I mutations resulted in constitutive expression regardless of the presence of thiamin, while Class II mutations resulted in constitutive repression. In vitro methods were used to determine the effect of these nucleotide changes on TPP-dependent conformational changes at the SD region. Results from these experiments suggest that the SD region of Class I mutant constructs is accessible regardless of the presence of TPP. However, in Class II mutant constructs, the RNA favors a closed conformation in which accessibility of the SD region is limited, resulting in inhibition of translation regardless of the presence of TPP.

Keywords: THI-box, riboswitch, regulation

59. Mechanism of Editing of Mis-charged Cys-tRNAPro by Bacterial YbaK

Byung Ran So (Chemistry, Ohio State University), Meredith Qualley (Chemistry, Ohio State University), Karin Musier-Forsyth (Chemistry and Biochemistry, Ohio State University)

Abstract:
Aminoacyl-tRNA synthetases are essential enzymes that help to ensure the fidelity of protein translation by accurately aminoacylating (or “charging”) specific tRNA substrates with cognate amino acids. Many synthetases have an additional catalytic activity to confer amino acid editing or proofreading. This activity relieves ambiguities during translation of the genetic code that result from one synthetase activating multiple amino acid substrates. For example, prolyl-tRNA synthetase (ProRS) mis-activates alanine and deacylates mischarged Ala-tRNAPro using an editing active site that is distinct from the site of amino acid activation. A free-standing protein (YbaK) with homology to the ProRS editing domain is present in most bacteria. YbaK has been shown to possess hydrolytic editing activity against mischarged Cys-tRNAPro. Previously, we demonstrated that the strictly conserved K46 residue in the putative substrate-binding pocket is critical for Cys-tRNAPro editing activity, and that the specificity of trans-editing by YbaK is ensured through formation of a novel ProRS/YbaK/tRNA ternary complex. To further characterize the trans-editing mechanism of YbaK, we performed extensive Ala-scanning mutagenesis of conserved residues, as well as residues identified from substrate docking studies and molecular dynamics simulations. Fluorescence anisotropy and isothermal titration calorimetry binding experiments were also performed to probe ternary complex formation with homologous ProRS and tRNA species. Taken together, the results of these studies allow us to propose a mechanism of catalysis by YbaK involving conserved residues within the substrate-binding region, and to begin to define the stoichiometry and interactions between the three binding partners.

Keywords: Prolyl-tRNA synthetase, YbaK

60. Characterizing the essential role of Fap7 ATPase activity in yeast ribosome assembly

Bethany Strunk (Chemical Biology , University of Michigan), Heather Woolls (Chemical Biology, University of Michigan), Jamie Van Etten (Chemical Biology, University of Michigan), Devon Riter (Department of Chemistry, University of Michigan), Katrin Karbstein (Department of Chemistry, University of Michigan)

Abstract:
In Saccharomyces cerevisiae, the final cytoplasmic step in the maturation of 40S ribosomes involves cleavage of 20S rRNA to generate the mature 3´ end of 18S rRNA. Several accessory factors are involved in this step, mutations of which lead to accumulation of 20S rRNA in the cytoplasm and failure to produce mature, active ribosomes (1). One such factor is Fap7 an essential protein with ATPase sequence homology believed to act through transient association with the maturing small subunit (2). Conserved amino acids predicted to be involved in ATP hydrolysis are required for Fap7\'s 18S rRNA processing activity (2). We are using ATPase assays as well as detection of changes in intrinsic tryptophan fluorescence upon nucleotide binding as tools to investigate Fap7´s essential role in ribosome assembly. Preliminary data indicate that purified Fap7 has weak ATPase activity and that this activity may be regulated by reversible disulfide bond formation. Interestingly, Fap7 has also been implicated in regulating the transcriptional response to oxidative stress (3) and may thus integrate transcriptional and translational response to oxidative stress using disulfide bond formation as a sensor.

References:
1. Peng et al. Cell. 2003 113(7): 919-33.
2. Granneman et al. Mol Cell Biol. 2005 25(23): 10352-64.
3. Juhnke et al. Mol Microbiol. 2000 35(4): 936-48.

Keywords: rRNA processing, yeast, Fap7

61. Investigating the Interaction between HTLV-2 Matrix and its RNA Packaging Signal

Meng Sun (Department of Chemistry, The Ohio State University), Karin Musier-Forsyth (Department of Chemistry, The Ohio State University)

Abstract:
Retroviral RNA encapsidation involves a recognition event between genomic RNA packaging signals and one or more domains in Gag. The primary packaging signal of deltaretroviruses including BLV, HTLV-1 and HTLV-2 was predicted to be two stem-loop structures, SL1 and SL2, which are located in the 5-UTR region. Previous work showed that mutation of conserved charged residues in the BLV matrix (MA) domain affects virus replication and viral RNA packaging efficiencies. To gain further insight into the role of the MA domain of Gag in directing viral RNA packaging, we examined the interaction of HTLV-2 MA and its putative packaging signal in vitro. We also used mutagenesis to probe the role of conserved charged amino acid residues of MA in aggregating and binding non-specific and specific nucleic acids. Electrophoretic mobility shift assays and fluorescence anisotropy measurements were performed to obtain the binding affinity of MA and its mutants to nucleic acids. In general, HTLV-2 MA displays higher binding affinity to nucleic acids than either HIV-1 MA or HTLV-1 NC. The simultaneous mutation of two basic residues (R47A/K51A) in MA á-helix II, results in a severe binding defect relative to wild-type, whereas single point mutations have more modest effects on binding. In addition, MA binds with similar affinity to single- or double-stranded nucleic acids or stem-loop structures. Taken together, these results are consistent with a direct interaction between HTLV-2 MA and its packaging signal, and this interaction is driven, at least in part, by electrostatic forces.

Keywords: Matrix, packaging, interaction

62. The TSC1 gene is Regulation by microRNAs

Apana AghaL. Takwi (Department of Biochemistry and Molecular Biology, School of Medicine, University of Louisville)

Abstract:
Tuberous sclerosis complex TSC) is a human genetic syndrome forming benign tumors in the brain, kidney, eyes, heart and skin affecting an estimate of 1 in every 6000 people. TSC is caused mainly by the lost of tuberin (TSC2) and/or Harmatin (TSC1) proteins, two tumor suppressor genes. TSC1 binds and stabilizes TSC2 and prevents its potential ubiquitination and degradation. The TSC1/TSC2 heterodimer has been placed under the phosphatidylinositol 3-OH kinase (PI-3K)-mammalian target of rapamycin (mTOR)/S6K1/4E-BP1 signaling pathway. It functions by inhibiting mTOR activity through a small GTPase RHEB. The TSC2 gene has a very short 3’-UTR (150bp), while TSC1 has a very large 3’-UTR (4887bp). The observation led us to hypothesize that one possible cause of TSC is that microRNAs dysregulates TSC1 gene expression. MicroRNAs (miRNA) are 20-22bp short RNAs that regulate gene expression by binding to their target gene’s mRNA 3’-UTR and either lead to their degradation or repression of translation. Numerous databases have been set up that are used to predict miRNA that might target a given gene. From one such database, www.microma.sanger.ac.uk, we found 31 miRNA that were predicted to target TSC1. We cloned the TSC1 3’-UTR into phRL-TK vector and performed a luciferase reporter assay. Our results indicate that a total of 14 miRNAs (miR- 361, 328, 126, 32, 200a, 326, 19b, 19a, 345, 301, 130b, 214, 523 and let-7e) reduced luciferase gene expression by more than 25%. Western blots analysis using 10 of these miRNA, transfected into both SV7tert and 293T cells, indicated that miR-130b, 200a, 32, and 126 effectively down-regulated the TSC1 gene. Although much work and further confirmatory experiments are needed, the present results suggest that TSC1 is targeted by miRNAs.

Keywords: Tuberous sclerosis complex, microRNA, Luciferase assay

63. Embryonic Expression Patterns Implicate Muscleblind-like Proteins in Cardiac Morphogenesis

Fulvia Terenzi (Department of Cell Biology, Lerner Research Institute, Cleveland Clinic), Kyle R. Brimacombe (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 muscle-like (MBNL) family have been shown to regulate pre-mRNA alternative splicing, and have been implicated in developmental-stage specific splicing in heart and skeletal muscle during the fetal-to-adult transition. To investigate the role of MBNL proteins during embryonic cardiogenesis, we examined MBNL expression in the developing chicken heart. By whole mount in situ hybridization, MBNL1 and MBNL2 were first detected in the embryonic heart at 48 hours (H&H stage 14), several hours after the primitive heart tube has formed, when cardiac looping is well underway. Expression continues in the heart during subsequent cardiac morphogenesis, as endocardial cushion outgrowth septates the heart into chambers (H&H stages 18-23). In situ hybridization on sections of H&H stage 23 embryos reveals strong MBNL1 and MBNL2 expression in the myocardium and in endocardial cells within the endocardial cushions, but not within the endocardium in other regions of the heart. MBNL3 is not detected in the heart at any stage. MBNL1 and MBNL2 are themselves alternatively spliced, and undergo changes in alternative splicing in the heart during endocardial cushion induction, outgrowth, and remodeling (H&H stages 14-29). Consistent with this, western blot analyses on total protein samples from whole hearts revealed that multiple MBNL1 protein isoforms are expressed in the embryonic heart, and the expression of these isoforms is dynamic during endocardial cushion development. These changes in MBNL expression coincide with a change in the pattern of cardiac troponin T (cTNT) alternative splicing, a known pre-mRNA target of MBNL activity. Together, these results suggest that MBNL proteins not only participate in fetal-to-adult heart development, but may also influence developmentally regulated splicing events during embryonic cardiac morphogenesis.

Keywords: alternative splicing, muscleblind-like, development

64. PKR Activation by Stem-loop RNAs is 5’-Triphosphate Dependent

Subba Rao Nallagatla (Department of Chemistry, The Pennsylvania State University), Jungwook Hwang (Integrative Biosciences (IBIOS), The Pennsylvania State University), Rebecca Toroney (Department of Chemistry, The Pennsylvania State University), Xiaofeng Zheng (Department of Biochemistry and Molecular Biology, Life Sciences College, Peking University), Craig, E. Cameron (Integrative Biosciences (IBIOS) and Department of Biochemistry and Molecular Biology, The Pennsylvania State University), Philip C. Bevilacqua (Department of Chemistry, The Pennsylvania State University)

Abstract:
The protein kinase PKR is an interferon-induced enzyme in the innate immune system. In response to foreign RNA, PKR undergoes autophosphorylation and phosphorylates eIF2?, which inhibits translation initiation. While long stretches of double-stranded RNA activate PKR, certain transcripts containing imperfections and single-stranded regions can also activate it. We report that single-stranded (ss) RNAs with limited secondary structure activate PKR in a 5’-triphosphate-dependent fashion in vitro and in vivo. Moreover, ssRNAs with cellular 5’-end signatures, such as 7-methyl-guanosine or monophosphate, do not activate PKR. We demonstrate that PKR activation works independent of RIG-I and that interferon-± treatment enhances PKR’s ability to sense 5’-triphoshorylated ssRNA. These findings suggest that PKR surveillance of molecular features at the 5’-end of RNA presents a checkpoint in innate immunity.

Keywords: PKR, 5-triphosphate

65. The T. brucei mitochondrial desulfurase IscS is essential for tRNA thiolation: implications for tRNA editing and import

Jessica Wohlgamuth-Benedum (Department of Microbiology, The Ohio State University), Julius Lukes (Institute of Parasitology, Czech Academy of Sciences), Juan D. Alfonzo (Department of Microbiology, The Ohio State University)

Abstract:
In Leishmania and Trypanosoma species, tRNAs are transcribed in the nucleus and imported from the cytosol into the mitochondrion. The only tRNATrp encoded within the nucleus of these organisms contains a CCA anticodon that can decode the UGG codon in the cytosol, but is unable to decode the UGA tryptophan codons in mitochondria. Trypanosomatids have solved this problem of UGA decoding by specifically changing the first position of the anticodon of tRNATrpCCA from cytidine (C34) to uridine (U34) by RNA editing, following import into the mitochondrion. In addition, this tRNA undergoes a number of posttranscriptional chemical modifications in the anticodon stem loop following mitochondrial import. Notably, position 33 (adjacent to the edited nucleotide) becomes thiolated but only in the edited tRNA species. We have hypothesized that thiolation of the normally unmodified U33 (97% of all the sequenced tRNAs from various organisms have an unmodified U33) to be an essential prerequisite for C to U editing of tRNATrp in trypanosomatids and thus important for cell viability. In E. coli, formation of s2U in tRNA is mediated by the cysteine desulfurase, IscS. In Trypanosoma brucei there are two IscS homologs, a cytosolic protein and a protein targeted to the mitochondria. Here we show by using RNAi that the mitochondria-targeted IscS is responsible for s2U formation in both cytosolic and mitochondrial tRNAs. Since thiolation of tRNAs may play a critical role in both tRNA editing and import into the mitochondria, further studies should shed light into how this modification may regulate both processes.

Keywords: RNA, Modification

66. Gene Prediction in Physarum and Didymium Mitochondria by Comparative Approach for RNA Editing

Wei Zhang (Biophysics program, The Ohio State University), Ralf Bundschuh (Department of Physics and Biophysics program, The Ohio State University)

Abstract:
Mitochondrial gene identification in Physarum polycephalum and Didymium iridis is a complex process because of frequent RNA editing in which nucleotides are inserted, deleted or substituted during DNA transcription so that DNA and mRNA sequences for the same gene are different. Comparison of two genomes has proved to be a powerful approach to solve this. Alignment results by NCBI bl2seq software, based on complete Physarum and partial Didymium mitochondrial sequences, show 42 conserved regions. 13 of such regions match 2 known rRNAs in Physarum, indicating 2 rRNAs in Didymium. 14 match 11 known protein coding genes in Physarum, indicating 8 unknown genes and confirming 3 known ones in Didymium. 11 indicate other 10 protein coding genes in both organisms and are also in accordance with the results previously predicted by our existing program PIE that only works on Physarum. 3 are newly found and their details are unknown so far. The remaining 1 is believed to be tRNA because it is short and close to rRNAs. With the remaining part of Didymium genome to have been sequenced, this work will help identify more unknown genes and discover the editing mechanisms in both organisms.

Keywords: comparative approach, editing mechanism

67. RNA Folding Dynamics by Single Molecule Temperature Jump Kinetics

Rui Zhao (Wayne State University), Elvin Aleman (Wayne State University), Rueda (Wayne State University)

Abstract:
RNA molecules play a vital role in numerous cellular processes, such as protein synthesis, RNA splicing and control of gene expression. In order ot become active RNA molecules must first fold into their native structure. However, the RNA folding problem remains largely unsolved impeding progress in understanding the structure-function relationship in biologically important RNAs, and in engineering RNA molecules with useful applications in a wide variety of fields such as life-sciences, nanotechnology, etc. Single molecule spectroscopy is a very powerful tool to study the RNA folding problem because single molecule trajectories uncover key information otherwise hidden in ensemble-averaged bulk experiments.

RNA folding energy surfaces are usually rough and may contain kinetic traps that can form biologically inactive intermediates. In order to investigate the folding energy surface of RNA molecules, we have developed new technology based on the combination of single molecule spectroscopy with infrared laser induced temperature jump kinetics (single molecule T-jump). Single molecule T-jump kinetics enable us to monitor the folding dynamics of single RNA molecules in real time, and to characterize their folding landscape. We will present preliminary data of single molecule T-jump kinetic time trajectories applied to DsrA RNA. This technique will be readily adapted to study the folding of biologically important RNA enzymes, as well as DNA and proteins.

References:
D. Rueda and N.Walter, J.Nanoscience and Nanotechnology(2005)
DJ Proctor, H Ma, E Kierzek, R Kierzek, M Gruebel, Biochemistry, 2004
H Ma, DJ Proctor, E Kierzek, R Kierzek, PC J. Am. Chem. Soc, 2006
Kolk, M.H., Heus, H.A. and Hilbers, C.W. (1997) EMBO J., 16, 3685-3692
http://www.bioinfo.rpi.edu/applications/mfold/rna/form1.cgi

Keywords: Single Molecule, T-jump, kinetics

68. Azasugar Affinity for T Box Antiterminator Model RNA: Potential new class of RNA ligands

Shu Zhou (Department of Chemistry and Biochemistry, Ohio University), Samira Telshow (Department of Chemistry and Mineralogy, Leipzig University, Germany), Dr. Jennifer V. Hines (Department of Chemistry and Biochemistry, Ohio University)

Abstract:
The T box genes, which are found in Gram-positive bacteria, contain a highly-conserved sequence of nucleotides known as the T box sequence. Expression of these genes is regulated by the interaction of uncharged cognate tRNA with the 5’ untranslated region of the mRNA during transcription. This interaction involves, in part, the base pairing of the tRNA accepter end with four bases of the antiterminator, which prevents the formation of an alternative terminator secondary structure and results in complete transcription of the gene. In an effort to investigate the potential for molecular modulation of this mechanism with exogenous small molecules a set of azasugar ligands were investigated. The azasugar class of ligands are poly-hydroxylated nitrogen heterocycles. These carbohydrate analogs can be protonated at physiological pH resulting in chemical characteristics similar to aminoglycosides. Given the ability of T box antiterminator model RNA to bind aminoglyocosides, it was hypothesized that the antiterminator may also bind azasugars. Enzymatic probing, circular dichroism and fluorescence-based ligand binding assays were conducted to investigate the affinity and RNA structural effects of azasugar ligands binding to wild-type and a reduced function variant antiterminator model RNA .

Keywords: T box tranascription antitermination, Ligand, Fluorescence

69. Application of RNA nanotechnology in gene delivery and therapy

Songchuan Guo (Department of Comparative Pathobiology and Department of Biomedical Engineering, Purdue Univeristy), Yi Shu (Department of Biomedical Engineering, University of Cincinnati), Steve Perrin (Department of Biomedical Engineering, University of Cincinnati), Hongkwan Cho (Department of Biomedical Engineering, University of Cincinnati), Jing Liu (Department of Biomedical Engineering, University of Cincinnati), Peixuan Guo (Department of Comparative Pathobiology and Department of Biomedical Engineering, Purdue Univeristy, Department of Biomedical Engineering, University of Cincinnati)

Abstract:
By utilizing RNA nanotechnology, we engineered both therapeutic siRNA and a receptor-binding RNA aptamer into individual pRNAs of phi29's motor. The RNA building block harboring siRNA or other therapeutic molecules was fabricated subsequently into a trimer through the interaction of engineered right and left interlocking RNA loops. The incubation of the protein free nanoscale particles containing the receptor-binding aptamer or other ligands resulted in the binding and co-entry of the trivalent therapeutic particles into cells, subsequently modulating the apoptosis of cancer cells and leukemia model lymphocytes in cell culture and animal trials. The use of such antigenicity-free 20-nm particles holds promise for the repeated long-term treatment of chronic diseases.

Keywords: phi29 motor, pRNA, siRNA