RRM
2007Rustbelt RNA Meeting
RRM
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Talk abstracts
Abstract:
In mammalian cells, pre-mRNA splicing factors and other pre-mRNA processing factors are stored in nuclear bodies called nuclear speckles. Proteomic analysis of nuclear speckles reflected enrichment of splicing factors in nuclear speckles and revealed 33 novel speckle proteins including Son (Saitoh, N., C. S. Spahr, S. Patterson, P. Bubulya, A. F. Neuwald and D. L. Spector. 2004. Proteomic analysis of interchromatin granule clusters. Mol. Biol. Cell. 15:3876-3890). Son is a large protein (2564 amino acid length) that contains six types of repetitive sequences that cover approximately one-third of its sequence and that are not found in any other protein. This study aims to determine nuclear functions for Son in pre-mRNA transcription and splicing as well as in structural organization of speckles.
We have recently generated rabbit polyclonal antibodies against both the amino- and the carboxy-termini of Son. Immunofluorescence localization of endogenous Son with these antibodies shows colocalization of Son with splicing factor 2/alternative splicing factor (SF2/ASF) in nuclear speckles. In addition, we fused Son with yellow fluorescent protein (YFP) and stably expressed YFP-Son in Hela cells where it also co-localizes with SF2/ASF in nuclear speckles. Interestingly, Hela cells that are treated with Son siRNA oligos show a complete reorganization of nuclear speckle components (including SR proteins and snRNP proteins) from their typical irregular shape into a donut-like shape. The altered organization of nuclear speckles observed after Son depletion implicates Son in a novel and essential role in the structural maintenance of nuclear speckles. Ongoing studies will elucidate the recruitment of Son to gene loci, and will determine the domains of Son required for nuclear speckle integrity and gene regulation.
Keywords: nuclear speckles, gene expression
Abstract:
DEAD-box proteins rearrange RNA and RNP structures in an ATP-dependent fashion, but it is unknown how ATP binding and hydrolysis are coupled to the remodeling processes. Here we have analyzed the coupling of RNA duplex unwinding to ATP binding and hydrolysis by the DEAD-box protein Ded1p from Saccharomyces cerevisiae. We show that the actual strand separation during duplex unwinding requires only ATP binding but not actual hydrolysis. ATP hydrolysis is used to enable the enzyme to dissociate from the RNA substrate subsequent to the strand separation, which is important for substrate turnover. Our data show that no coupling exists between the energy released during ATP hydrolysis and strand separation by Ded1p. The findings explain mechanistic features of DEAD-box proteins that previously have been difficult to interpret.
Keywords: ATP hydrolysis, unwinding, DEAD-box
Abstract:
The regulated turnover of mRNA is recognized as a vital aspect of gene expression. The importance of maintaining appropriate mRNA decay is exemplified by the complexity of the decay process; there are three distinct steps (deadenylation, decapping, and exonucleolytic decay) and over 20 protein factors are involved. An additional layer of complexity is manifested by the observation that the process of mRNA degradation is intertwined with mRNA translation, exhibiting an inverse relationship. Specifically, decrease in the rate translational initiation lead to dramatic destabilization of mRNAs. Additionally, translational initiation defects can be suppressed by mutations of a component of the mRNA decapping factor. Conversely, inhibition of mRNA translation elongation, either in cis or in trans, results in a dramatic stabilization of mRNAs. This has lead to a well accepted model in which the cessation of mRNA translation is thought to be an initial and necessary step in the regulated destruction of cytoplasmic mRNA transcripts. Despite these long-standing observations, the precise and detailed mechanism of how mRNA translation and mRNA decay are coupled remains ambiguous.
Our work focuses on dissecting the complex interconnectivity between mRNA decay and mRNA translation. Recent data have pointed to the observation that mRNA decay specifically impacts the process of translational elongation. First, we find that mutation in factors involved in the second step of mRNA decay, i.e. the decapping reaction, are extraordinarily sensitivity to drugs that inhibit the process of translational elongation. Consistent with this, mRNAs accumulate with an abnormally large number of bound ribosomes in decapping mutants when compared to wild-type cells. These results suggest that the rate of translation elongation is slowed in these mutants. Consistent with this, direct measurement of the ribosome transit time in decapping mutants reveals that translation elongation is compromised when decapping does not occur appropriately. We are currently in the process of determining how decapping of mRNA is connected to translational elongation, but believe, however, that investigation into this newly recognized step of gene regulation will have significant impact on our overall comprehension of the relationship between mRNA translation and turn-over and in understanding the regulation of gene expression in a variety of biological contexts.
Keywords: mRNA turnover, translation elongation, decapping
Abstract:
MicroRNAs (miRNA) are short 20-25 nucleotide RNA molecules that negatively regulate gene expression in animals and plants. Growing evidence indicates that microRNAs (miRNAs) are key participants in the cellular signaling pathway regulatory network. We have constructed the first lentiviral-based microRNA genetic library that consists of most human miRNA minigenes. Our modification of a third-generation Feline Immunodeficiency Virus (FIV)-based vector provides two genetic markers, neomycin and GFP, allowing both transient observation and stable selection. The lentiviral vector will also allow one to use packaged pseudoviral particles to transduce hard-to-transfect cells. We have utilized this genetic library to study NF-kB signaling pathways using a dual-reporter assay and identified a large number of microRNAs modulating NF-kB signaling. Using Western blotting and other analyses, we determined some microRNA targets in the canonical NF-kB signaling pathway. The present work reveals that microRNAs mediate critical signaling pathways, demonstrating added complexity of human signaling networks.
Keywords: microRNA, NF-kB signaling pathway, lentiviral-based genetic library
Abstract:
Schizophrenia (SZ) is a severe neuropsychiatric illness affecting 1% of the adult population, costing the U.S. economy in excess of $60 Billion annually. Decades of SZ study has shown that detectable variations in coding genes are scarce. We hypothesize that a microRNA is a SZ-susceptibility gene. A microRNA (miRNA) regulates its target gene expression by inhibiting translation initiation or degrading the mRNA. By predicting the gene-targeting ratio between the SZ genes as the target pool and the whole genome as the target pool, we identified a miRNA targeting SZ genes twice more frequently than expected. The frequencies for other miRNAs are largely comparable, i.e. the frequency for a miRNA targeting the whole genome as a pool tends to match that of SZ genes as a pool. This miRNA gene, hsa-mir-346, is located in the intron 2 region of a linkage mapped SZ-susceptibility gene, Grid1. Total RNA was extracted from Brodmann's area 46 of the dorsolateral prefrontal cortex from SZ and bipolar patients, as well as normal control subjects. Using real-time PCR, we determined the expression level of the miRNA in the total RNA samples and found that the miRNA expression level is 42% lower (p= 0.02) in SZ patients and 32% lower (p=0.09) in bipolar patients compared to normal subjects. The host gene expression level is roughly colinear with that of the miRNA, but with a lower confidence level. This study demonstrates for the first time that a quantifiable genetic distinction exists between the schizophrenia and the normal subjects and that the etiology of a linkage mapped SZ susceptibility locus involves a miRNA gene which potentially has far-reaching regulatory consequences.
Keywords: schizophrenia, bipolar, microRNA hsa-mir-346, real-time PCR
Abstract:
Transfer RNAs (tRNA) containing inosine (I) at the first, or "wobble" position of the anticodon have been observed in two of the three domains of life and when present is essential for cell viability. Inosine formation at the wobble position expands the decoding capabilities of a tRNA, as inosine can base pair with adenosine (A), cytidine (C), or uridine (U), allowing the decoding of multiple codons by a single tRNA. The adenosine deaminases acting on tRNA (ADATs) in bacteria (ADATa) are homodimeric enzymes that recognize only one substrate. In eukarya, the enzyme is a heterodimer composed of two sub-units, ADAT2 and ADAT3 that can recognize seven different tRNAs as substrates. ADAT2 is the catalytically active sub-unit while ADAT3 contains a proposed pseudo-active site but may not be directly responsible for deamination. While the tRNA editing mechanism has been elucidated, the precise residues that provide substrate binding to the enzyme are not known. Here we show the contribution of individual residues in ADAT2 and ADAT3 from Trypanosoma brucei in both the ability to catalyze the deamination reaction and the ability to recognize the tRNA substrate. Mutations to the active site and pseudo active site of the editing complex have catalytic effects although the mutant complex is not deficient in binding tRNA. Deletion of a stretch of charged amino acids at the c-terminus of ADAT2 abolishes both the binding and the deamination activity of the editing complex. Our results provide a first glance of the key residues involved in tRNA binding by this group of editing enzymes.
Keywords: RNA Editing, Deaminase
Abstract:
During translation, tRNAs must move rapidly to their adjacent sites in the ribosome while maintaining precise pairing with the mRNA. A growing body of evidence suggests that this movement (translocation) occurs in a stepwise manner with hybrid-state intermediates, but it is unclear how these intermediate binding states relate to the kinetically defined events of translocation. Here we employ ribosomes harboring mutations of E-site rRNA nucleotide C2394 in a pre-steady-state kinetic analysis of translocation. These mutations are predicted to inhibit P/E state formation based on structural studies. Each substitution at position 2394 decreases growth rate, the maximal rate of translocation (ktrans), and the apparent affinity of EF-G for the pretranslocation complex (K1/2). The magnitude of these defects follows the trend (A > G > U > WT). The most deleterious mutation, C2394A, does not affect the rate of GTP hydrolysis. Thus, the substantial (>20-fold) increase in K1/2 cannot be attributed to either the initial binding of EF-G or the subsequent GTP hydrolysis step. We propose that P/E state formation occurs subsequent to GTP hydrolysis and contributes to K1/2 because each of the first three steps of translocation is readily reversible. Substitution of the peptidyl group for an aminoacyl group, which is predicted to inhibit A/P state formation, has a different effect on translocation. The aminoacyl group decreases ktrans but has no effect on K1/2. These data suggest that movement of tRNA into the P/E and A/P states are separable events and the latter is effectively irreversible in the presence of EF-G.
Keywords: ribosome, translocation, tRNA
Abstract:
In all organisms precursor tRNAs are processed into mature functional units by post-transcriptional changes. These involve 5’ and 3’ end trimming as well as the addition of a significant number of chemical modifications, including RNA editing. The only known example of non-organellar C to U editing of tRNAs occurs in trypanosomatids. In this system, editing at position 32 of the anticodon loop of tRNAThr(AGU) stimulates, but is not required for, the subsequent formation of inosine at position 34. In the present work we expand the number of C to U edited tRNAs to include all the threonyl tRNA isoacceptors. Notably, the absence of a naturally encoded adenosine, at position 34, in two of these isoacceptors demonstrates that A to I is not required for C to U editing. We also show that C to U editing is a nuclear event while A to I is cytoplasmic, where C to U editing at position 32 occurs in the precursor tRNA prior to 5\' leader removal. Our data supports the view that C to U editing is more widespread than previously thought and is part of a stepwise process in the maturation of tRNAs in these organisms.
Keywords: editing, deamination, tRNA
Abstract:
tRNA is the most heavily modified tRNA in terms of the percentage of bases and has the most chemical diversity. The function of some of these modifications is still unknown. It has been suggested that some of them might play a role as sensors of enviromental stressors, but only this has only been demonstrated to be true for 4-thiouridine, which is a sensor for Near-UV light. There are links between oxidative stress responses and Near-UV light responses. To investigate the role of tRNA modifications, such as 4-thiouridine, in oxidative stress response, we have determined the levels of the tRNA modified bases at different time points and two different methods of inducing oxidative stress in E. coli
We find that hydrogen peroxide, a strong oxidant, and diamide, only a thiol oxidant, both cause changes in the tRNA modification patterns, that these patterns are unique, and that changes in the levels of the tRNA modifications occur only after 5 minutes of exposure and before any detectable growth defects. Analysis of mutants defecient in some tRNA modifications show different patterns than wild-type when exposed to oxidative stress.
Keywords: tRNA modification, oxidative stress
Abstract:
The tRNAHis guanylyltransferase (Thg1) catalyzes the addition of a single essential G-1 residue to the 5' end of tRNAHis, a modification that is universally conserved, is unique to histidyl-tRNA species, and is required for aminoacylation of tRNAHis in S. cerevisiae. Remarkably, Thg1 also catalyzes the template-dependent addition of multiple nucleotides in the 3'-5' direction to altered tRNA substrates; several features of this 3'-5' polymerization activity serve to distinguish it from the previously known physiological activity of Thg1, G-1 addition (Jackman & Phizicky, 2006). Since the reaction catalyzed by Thg1 is inherently unique, resulting in polymerization of nucleotides in the opposite direction of all other known DNA and RNA polymerases, and since the biological role of this unusual 3'-5' polymerization reaction remains a mystery, we have undertaken an investigation of the catalytic mechanism of nucleotide addition by Thg1, and of the substrate recognition requirements for both Thg1 activities, with the goal of defining optimal substrates for 3'-5' polymerization that could aid in the identification of in vivo substrates for this activity. Currently, we are investigating a collection of Thg1 variant proteins with alterations at highly conserved amino acids to define residues that play critical roles in G-1 addition, 3'-5' polymerization, or both activities. We are also examining the ability of Thg1 to catalyze 3'-5' polymerization with small DNA substrates, so as to consider the possibility of a previously unknown role for Thg1 in the repair or metabolism of DNA, in addition to its well-defined role in tRNA maturation.
References:
Jackman JE, Phizicky EM. 2006. tRNAHis guanylyltransferase catalyzes a 3'-5' polymerization reaction that is distinct from G-1 addition. Proc Natl Acad Sci U S A 103:8640-8645.
Keywords: Thg1, tRNAHis, 3-5 polymerase
Abstract:
Limited by the spatial resolution of optical microscopy, direct detection or counting of single components in biological complexes or nanoparticles is challenging, especially for RNA, which is conformationally versatile and structurally flexible. We report here the assembly of a customized single-molecule dual-viewing total internal reflection fluorescence imaging system for direct counting of RNA building blocks. The RNA molecules were labeled with a single fluorophore by in vitro transcription in the presence of a fluorescent AMP. Precise calculation of identical or mixed pRNA building blocks of one, two, three, or six copies within the bacteriophage phi29 DNA packaging motor or other complexes was demonstrated by applying a photobleaching assay and evaluated by binomial distribution. The dual-viewing system for excitation and recording at different wavelengths simultaneously will enable the differentiation of different complexes with different labels or relative motion of each labeled component in motion machines.
References:
Zhang H, Shu D, Huang F, and Guo P, Instrumentation and metrology for single RNA counting in biological complexes or nanoparticles by a single-molecule dual-view system, RNA 2007, in press.
Keywords: bacteriophage phi29 motor pRNA, single or dual labeling, viral DNA packaging motor
Abstract:
Bacterial virus phi29 DNA-packaging motor includes a connector geared by six pRNAs (packaging RNA) and a DNA packaging enzyme gp16. phi29 pRNA, with a minimum size of 117 bases, is another essential component of the motor. It contains two functional domains: the central procapsid binding domain and the 5'/3' ends paired the DNA translocation domain. pRNA forms dimers, trimers and hexamers via a hand-in-hand interaction through the base-pairing of two interlocking left- and right-hand loops. We designed a new artificial RNA molecule Y, bearing a sequence completely different from that of wild-type phi29 pRNA, can fold into the same secondary structure as that of pRNA. This RNA bound to the procapsid with an affinity similar to the wild type phi29 pRNA and was biologically active in the assembly of infectious phi29 virion. However, if 2-3 nucleotides on the right/left loops of artificial RNA Y were mutated, it resulted in the loss of activity in phi29 virion assembly. Further inverstigation of the single molecule photobleaching study on the procapsid/Cy3-artificial RNA Y complex revealed a good photobleaching pattern corresponding to the model of ring with six copies of pRNA. The principle for the design and construction of an artificial pRNA biologically active during a viral DNA packaging motor will be presented.
Keywords: Bacteriophage phi29, pRNA
Abstract:
All linear dsDNA viruses, including bacteriophage phi29, translocate their genome into a pre-assembled procapsid to near-crystalline density. The packaging enzyme in the viral DNA-packaging motor generates the driving force to overcome this entropically unfavorable packaging reaction. We investigated the specific interaction of the putative packaging enzyme of the phi29 packaging motor with packaging RNA (pRNA).
Our biochemical studies shows that the 5'/3' paired helical region on pRNA in the pRNA/procapsid complex serves as a docking site for gp16 binding. Gp16 bound to the pRNA-containing procapsid much more strongly than to the pRNA-free procapsid. The pRNA bulge C18C19A20 that is essential for DNA packaging was found to be dispensable for gp16 binding. These results imply that pRNA, with its central domain binds to the procapsid, extends its 5'/3' DNA-packaging domain for gp16 interaction. The interaction of gp16 with nucleic acids was also investigated using recently developed single molecule system. The technique employs TIRF enables to observe the binding between molecules. The study showed that gp16 binds nucleic acids (DNA or RNA) depending on their structures and chemistry. In addition, In vitro binding assay showed that gp16 binds to dsDNA in a non-sequence specific manner.
Gp16 contains a conserved nucleotide triphosphate binding motif. The steady-state analysis showed that the nucleic acids binding stimulates the ATP hydrolysis of gp16, and the extent is also dependent on the structure and chemistry of DNA or RNA. From the data, the kinetic parameters including Km and the rate constant for ATP hydrolysis were determined. Our results strongly suggest that gp16 interacts with nucleic acids including pRNA as a part of ATP hydrolyzing complex in the packaging motor, and its ATPase activity can be stimulated via those interactions.
Keywords: Bacteriophage phi29 packaing motor, Packaging RNA, Single molecule imaging
Abstract:
RNA-binding proteins Fox-1/Fox-2 were identified as the novel major neuron-specific negative regulators that mediate the neuron-specific calcitonin gene-related peptide (CGRP) processing pattern (Zhou et el, Mol Cell Biol 27, 830). Although it is clear that the regulatory effect of Fox proteins depends on two UGCAUG silencer RNA sequence motifs, it is not known how these proteins block inclusion of the calcitonin/CGRP of exon 4, which is an alternative 3’-terminal exon flanked by a 3’ splice site and a polyadenylation site on either end.
Here, we provide results of biochemical analysis to demonstrate that Fox-1/Fox-2 proteins directly inhibit splicing of calcitonin-specific exon 4 and blocks formation of the prespliceosomal early (E) complex by preventing the assembly of U2 snRNP auxiliary factor (U2AF) on the splicing acceptor site upstream of calcitonin-specific exon 4. Furthermore, using an MS2-MBP affinity purification approach, we demonstrated the mechanism whereby Fox proteins inhibit binding of U2AF. First, Fox proteins interfere with binding of the splicing factor 1 (SF1) to the branchpoint sequence through interacting with the -34 UGCAUG silencer element, which is immediately adjacent to the branchpoint. Second, binding of two SR proteins (Tra2-beta and SRp55) to exonic splicing enhancer elements is antagonized by the Fox proteins that interact with +45 UGCAUG silencer element. In HeLa cells, over-expression of Tra2-beta or SRp55 proteins can reverse the Fox-mediated reduction in calcitonin-specific exon 4 splicing pattern.
These results give the first mechanistic insights into how Fox proteins inhibit splicing, which remained unknown although alternative splicing of several pre-mRNAs has been shown to be regulated by these proteins.
Keywords: Fox protein, Calcitonin, splicing
Abstract:
Nuclear speckles are nuclear storage and assembly sites for pre-mRNA processing factors. Btf (Bcl-2-like transcription factor or BCLAF) was identified in a proteomic analysis of purified nuclear speckles and it localized in a unique pattern that resembles transcription sites(1). Our hypothesis is that Btf has a role in the coordination of transcription and co-transcriptional processing of pre-mRNAs. Recently reported cap-dependent association of Btf with in-vitro synthesized affinity purified MS2-AdML-M3 mRNPs is consistent with such a role(2). Additional evidence for Btf in transcription and/or splicing comes from our observations showing that Btf accumulates on a reporter gene locus in situ. Since Btf is enriched on the transcriptionally activated locus, but not on the inactive locus, we hypothesize that Btf is recruited to this locus during gene activation. To test this hypothesis, we are using this reporter locus to compare the timing of Btf recruitment with chromatin decondensation and the accumulation of chromatin remodeling factors, transcription factors and splicing factors.
SR splicing factors are typically targeted to nuclear speckles by RS domains. Although Btf has an arginine-serine-rich (RS) domain, it localizes predominantly around the periphery of nuclear speckles. We have performed deletion analysis to understand the unique Btf localization pattern. We have confirmed that the RS domain of Btf can function as a speckle targeting sequence on its own; however, removal of the Btf RS domain does not significantly change the Btf localization pattern. This suggests that Btf localization may be regulated differently than other speckle proteins. Our deletion analysis indicates that a specific region of Btf prevents its speckle localization, and that removing this region directs Btf to nuclear speckles. Ongoing studies aim to determine if this regulation occurs via post-translational modification of Btf, or by intermolecular interactions with binding partners.
References:
1.Saitoh, N. et al., 2004. Mol. Biol. Cell 15: 3876-3890.
2.Merz, C. et al., 2007. RNA. 13:1-13.
Keywords: splicing, Btf
Abstract:
The yeast SUV3 gene encodes a SKI2-like DExH helicase that is an essential component of the mitochondrial degradosome (mtEXO) that functions in RNA processing and turnover. Defects in SUV3 result in loss of respiration and molecular phenotypes associated with impaired RNA degradation, such as accumulation of excised introns and high-molecular-weight, unprocessed transcripts. A perplexing phenotype associated with inactivation of SUV3 is decreased splicing of the aI5&beta and bI3 group I introns. We have analyzed a weak allele of SUV3 that is respiratory incompetent, accumulates excised aI5&beta intron, and is defective in splicing of aI5&beta and bI3. Each of these phenotypes can be partially rescued by over expression of MRS1, which encodes a protein co-factor essential for the in vivo splicing of aI5&beta and bI3. In addition, we use an in vitro splicing assay to demonstrate that recombinant Suv3p protein can recycle recombinant Mrs1p protein from excised aI5&beta. These results suggest a model whereby Suv3p acts to promote Mrs1p dissociation, leading to the degradation of the excised aI5&beta intron. Collectively, this prevents the sequestration of Mrs1p and thereby promotes splicing of aI5&beta and bI3. Furthermore, this work has generated a novel system whereby the mechanisms by which a DExH helicase can displace RNA-bound proteins can be investigated in the context of a native RNP substrate.
Keywords: Helicase, Splicing, Turnover
Abstract:
MDM2, murine double-minute 2, and its related family member MDM4 are important for regulating the levels and activity of tumor suppressor protein, p53. Expression of alternatively spliced forms of these p53 regulators has been associated with various cancers. We have identified alternatively spliced forms of MDM2 and MDM4, which are induced after certain kinds of genotoxic stress.
Using MDM2 as a model, we are trying to understand the mechanism of alternative splicing of MDM2 and MDM4 in response to specific cell stress like UV exposure and cisplatin treatment. We have used minimal conserved genomic sequences from human and mice to create mini-gene constructs. These constructs contain the cis elements required to direct the alternative splicing in response to UV exposure in vivo. Using in vitro UV cross-linking with normal and cisplatin treated nuclear extract, we found factors that bind differently to our mini-gene constructs. Factors which bind to pre-mRNA of our mini-gene constructs in normal nuclear extract, but are displaced in the cisplatin treated nuclear extract may facilitate recognition of all MDM2 exons by the splicing machinery, and thus acts as a positive regulators of splicing. Likewise, we have identified specific proteins that bind to the MDM2 pre-mRNA only in the damaged extracts. These proteins are putative negative regulators of MDM2 splicing. These data underscore the dynamic nature of splicing machinery, which determines the splicing of not only MDM2 and MDM4, but also other genes. The UV-induced splicing machinery results from events initiated in response to stress and these RNA binding proteins may be the effectors of the damage-induced spliceome.
As opposed to a gene-specific approach to studying alternative splicing in cancer, this study will lead to a better understanding of how global signaling affects regulation of RNA processing factors involved in the generation of a damage-induced spliceome.
Keywords: MDM2, alternative splicing, spliceome
Abstract:
Previous work in our laboratory has shown that regulation of alternative splicing of Neurofibromatosis type I exon 23a is carried out by two groups of proteins: Hu proteins and TIA-1/TIAR proteins. Preliminary studies using different lengths of sequences upstream of NF1 exon 23a suggest that additional factors are involved in promoting skipping or inclusion of exon 23a. A series of GU-rich elements were identified in the sequence upstream of exon 23a, which led us to hypothesize that the CELF (CUGBP and ETR-3-like factors) family of RNA-binding proteins might regulate exon 23a alternative splicing.
We have investigated the role of CELF proteins in regulation of exon 23a inclusion using both cell model systems and transgenic mouse models. HeLa cells and CA77 cells, a rat neuron-like cell line, are excellent models because the endogenously expressed NF1 pre-mRNA is processed to predominantly include or skip exon 23a respectively in these cell lines. The expression level of CELF proteins correlates well with the splicing phenotype with HeLa cells expressing only one of the CELF proteins members while CA77 cells expressing all six members. Over-expression of CELF proteins in HeLa cells promoted skipping of exon 23a from a NF1 splicing reporter transcript. Additionally, siRNA knockdowns of endogenous CUG-BP and ETR-3, promote exon 23a inclusion in CA77 cells. Importantly, alternative splicing of NF1 exon 23a is also affected in mouse models that over-express CUG-BP1 in cardiac and skeletal muscles, indicating that the CELF functions observed in cell models are physiologically relevant. These results demonstrate that CELF proteins regulate tissue-specific skipping of the NF1 exon 23a. Future work will involve dissecting the mechanism by which CELF proteins regulate alternative splicing of NF1 exon 23a.
Keywords: CELF proteins, Neurofibromatosis Type I, Alternative Splicing
Abstract:
The T box antitermination system, which is located in the mRNA 5' untranslated leader region, is a tRNA-sensing riboswitch that exerts transcriptional control over many Gram-positive bacterial tRNA synthetase, amino acid biosynthesis, and amino acid transport genes (1). Control depends on the stabilization of a highly-conserved antiterminator structure via the binding of the gene's uncharged cognate tRNA (2). If the antiterminator is not stabilized, a competing terminator structure forms, halting transcription (3). The current investigations revolve around the structural features involved in the recognition of the tRNA acceptor stem by the antiterminator. Structural changes that occur in the antiterminator upon its binding with a microhelix tRNA model have been studied, allowing the derivation of a model for the binding of these two RNAs.
References:
1. Grundy, F. J. and Henkin, T. M. Crit. Rev. Biochem. Mol. Biol. 2006, 41, 329-338.
2. Grundy, F. J., et al. Nucleic Acids Res. 2002, 30, 1646-1655.
3. Grundy, F. J., et al. RNA, 2000, 30, 1065-1072.
Keywords: mRNA−tRNA interaction, riboswitch, fluorescence
Abstract:
The hairpin ribozyme is an RNA motif that facilitates cleavage and ligation of its own backbone. The catalytic activity and small size of the hairpin ribozyme makes it an attractive model system for understanding structure, dynamics, and function in RNA. Despite numerous structural and biochemical investigations the catalytic mechanism of the ribozyme’s self-cleavage remains unclear. However, multiple experiments do support the proposal that the protonation state of A38 plays a direct role in catalysis. In an attempt to gain insight into the impact of both A38 protonation and global stability on the catalytic activity of the hairpin ribozyme, we employ molecular dynamics (MD) simulations and native gel electrophoresis. We first examine the impact of an in vitro selected “gain of function” mutation at position 39 through both MD simulations and native gel electrophoresis. Our simulations predict improved global stability in this mutant which is validated by our observation that the mutant ribozyme migrates more rapidly during native gel electrophoresis. In order to investigate the impact of A38 protonation on active site architecture, we compare simulations in which A38 is either protonated or deprotonated at the N1 position. Our unprotonated simulations place N1 in close proximity to the nucleophilic 2’-O indicating that A38 may act as a general base in the cleavage reaction, a role that has generally been discounted based on proximities observed in crystal structures of the ribozyme in complex with a non-cleavable substrate analog. Simulations in which A38 is protonated place N1 in close proximity to the 5’-O leaving group which supports the proposal that A38 serves as a general acid in the cleavage reaction. Based on our simulations and previous biochemical and structural data we propose a new mechanism in which A38 acts as both general acid and general base.
Keywords: molecular dynamics, ribozyme, hairpin ribozyme
Abstract:
The 2’-OH is a unique character of RNA, and it affects RNA structure and function via the ribose conformation. Ribose conformation has previously been studied with 2’-OH modifications, such as methoxy (2’-OMe), fluorine (2’-F), and amino (2’-NH2) groups. Each modification has a preference of either C2’-endo or C3’-endo conformation; however, it is not 100%. Therefore, we use locked nucleic acid (LNA) to study the functional and thermodynamic effects of sugar conformation. LNA is typically used for antisense RNA chemistry and is locked completely into the C3’-endo conformation via a methylene linkage between 2’-O and 4’-C. We used well studied RNA structures (UUCG tetra loop and leadzyme) as a model. We tested the functional importance of sugar pucker dynamics in leadzyme catalysis. Structural and thermodynamic effects of ribose conformation are examined with the highly stable UUCG tetraloop. Finally, we are undertaking a preliminary analysis of LNA substitution effects on protein recognition of a structured RNA. As a result, LNA is useful as a probe of the functional and stability effects of structured RNA molecules.
Keywords: RNA catalysis, RNA structure, RNA-protein recognition
Abstract:
Recognition of cis-acting RNA elements by trans-acting RNA binding proteins is critical to the precise processing of RNA and for coordinated regulation of post-transcriptional gene expression. Traditional methods for mapping sites of RNA-protein interactions rely on in vitro equilibrium binding conditions focusing on single binding events. Here we describe a comprehensive method for mapping the in vivo sites of RNA-protein interactions across the human genome. We focused our initial studies on the shuttling SR protein SF2/ASF. This protein has well defined biochemical roles in post-transcriptional gene expression, however few bona fide endogenous targets have been identified. We employed cross-linking immunoprecipitation (CLIP) to purify covalent SF2/ASF-RNA complexes from HEK293T cells. A major advantage of CLIP is that photo cross-linking of intact living cells is used to capture the in situ specificity of RNA-protein interactions, without the added complications associated with chemical cross-linking (protein-protein cross-links). 45-55 nt RNA fragments corresponding to the in vivo binding sites for SF2/ASF were amplified and directly sequenced by ultra-high-throughput pyrosequencing using the 454 platform. More than 150,000 sequences encompassing 8,166 genomic regions were mapped to the human genome using BLAT. 4,481 genomic regions were represented by multiple amplicons, termed replicons. Approximately 50% of these regions mapped to non-repetitive genomic loci. These regions contain 1463 intragenic and 431 intergenic loci, respectively. We find that SF2/ASF binding sites are strongly biased towards exonic sequences and exon-intron boundaries relative to intronic positions (76% compared with 24%, respectively). We determined a mean PhastCons score for all 8,166 genomic regions identified by pyroCLIP. Genomic regions containing multiple amplicons sequences are highly conserved. Importantly, this correlation is observed for both intra- and intergenic binding sites for SF2/ASF, suggesting that SF2/ASF may be involved in processing of noncoding RNAs or novel protein coding genes. Finally clustering of SF2/ASF mRNA targets based upon primary encoded function reveals that there is a highly significant enrichment (p value = 10-74) for genes involved in mRNA metabolism. Roles for SF2/ASF in RNA processing will be discussed based upon this novel genome wide perspective.
Keywords: RNA binding protein, genomics, high throughput sequencing
Abstract:
We have developed an intermolecular system for domain docking in the hairpin ribozyme as a simple model for the formation of tertiary structure in RNA. Our ultimate goal is to map out the free energy surface for the conformational changes involved in tertiary structure formation using comparisons of the conformational dynamics in free and bound states of the individual loops. Toward this end, we report progress in the biophysical characterization of this interaction including isothermal titration calorimetry analysis of docking thermodynamics, an exploration of the role(s) of monovalent and multivalent cations in docking, and a preliminary analysis of docking kinetics using surface plasmon resonance. In many respects, our data are in gratifying agreement with earlier biochemical results on this system (Hampel, Walter, and Burke, Biochemistry 37, 14672-14682, 1998). The current results lay a strong biophysical foundation for future NMR studies of the docking transition.
Keywords: hairpin ribozyme, RNA tertiary structure, thermodynamics
