RRM
2010 Rustbelt RNA Meeting
RRM
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Talk abstracts
Abstract not available online - please check the printed booklet.
Abstract:
The results of large scale transcriptome analyses suggest that while over 93% of the human genome is transcribed into RNA, ORFs and their associated UTRs occupy only 2% of the genome. It is estimated that the human genome contains over 70,000 large non-protein coding transcripts (lncRNAs); however, many aspects of the biology and function of this novel class of cellular regulators are almost completely unknown.
We have analyzed a ~2700 nt lncRNA that is spliced and polyadenylated. Interestingly, we found that the expression of the lncRNA is induced when cells are exposed to stressful conditions in several different cell types of mesodermal and ectodermal origin. We analyzed the expression of factors that are involved in cellular stress response and apoptosis in cells that either overexpressed the lncRNA or in which the RNA was knocked down using shRNA-mediated strategies. After exposure to stress, the knockdown cells exhibited an impaired cellular stress response and a much higher death rate compared to controls, while the overexpression cells showed an elevated level of stress response and a cellular stress-resistant phenotype. Analysis of the stress response factors indicated that some but not all heat shock proteins had a higher basal level in the overexpression cells even in the absence of stress and thus, the elevated expression of the lncRNA seemed to “pre-condition” the cells for tolerance of stress. Interestingly, in the knockdown cells, the induction of heat shock proteins after stress was both delayed and reduced in magnitude. We made a series of truncation mutations in the lncRNA and could show that deletion of part of the RNA close to its 5' end abolished its ability to impart stress resistance in transfected cells, while the other mutants did not have this effect. Together, these data indicate that the lncRNA plays an important role in regulation of the stress response and cellular survival under stressful conditions and demonstrates the power of lncRNAs as a novel class of regulatory molecules in higher eukaryotes.
Keywords: lncRNA, Stress response
Abstract not available online - please check the printed booklet.
Abstract:
In this study we document that decapping-dependent turnover is the major pathway of decay for key non-coding RNAs (ncRNAs) generated from the GAL and PHO84 loci in budding yeast. Additionally, we show at the GAL locus, decapping of the regulatory ncRNA (GAL1 uCUT) is required for efficient expression of the GAL1 mRNA.
The role of the decapping enzyme in controlling cytoplasmic mRNA levels is well documented. In the cytoplasm, mRNA turnover is initiated by the loss of the poly(A) tail, followed by decapping, and subsequent 5’-3’ exoribonuclease digestion. The majority of RNA polymerase II transcripts receive a 5’ cap structure, and in recent years it has become clear that RNA polymerase II (pol II) transcribes more than just mRNAs. Indeed, transcription by pol II is pervasive with estimates of RNA transcripts originating from up to 80% of the non-repetitive sequence of the S. cerevisiae genome (1). Moreover, close examination of the transcriptional output of yeast cells has revealed the existence of cryptic unstable transcripts (CUTs) that are virtually undetectable in wild-type cells, but accumulate in the absence of the nuclear exosome (2). Here we document that the destruction of known CUTs are also controlled by the decapping enzyme. In a novel decapping pathway, decay of these CUTs is dependent on the catalytic activity of the decapping enzyme, but is independent of deadenylation as well as known decapping regulators. Additionally, we show that stabilization of the ncRNA by the loss of the decapping enzyme results in a severe impairment in neighboring gene expression. Furthermore, we show decapping-dependent decay of a key ncRNA influences the chromatin state of neighboring genes, thereby impacting the overall gene expression profile. These data highlight a novel cellular role for the mRNA decapping enzyme in impacting the overall architecture of the transcriptome.
References:
1. Nagalakshmi, U., et al., The transcriptional landscape of the yeast genome defined by RNA sequencing. Science, 2008. 320(5881): p. 1344-9.
2. Wyers, F., et al., Cryptic pol II transcripts are degraded by a nuclear quality control pathway involving a new poly(A) polymerase. Cell, 2005. 121(5): p. 725-37.
Keywords: non-coding RNA, decapping, histone modification
Abstract:
Mirtrons are a recently discovered class of microRNA that are generated by a nuclear processing pathway that is distinct from canonical miRNAs. Mirtrons are defined by their dependence on splicing and debranching of the excised intron lariat, rather than the miRNA microprocessor complex, for the production of the pre-miRNA. Mirtrons were first described in Drosophila and have been predicted, but are yet to be experimentally validated, in humans. To examine the processing of putative mirtrons in humans, we constructed a number of minigenes comprised of a mirtron-containing intron and the flanking exons. We found that production of some of the predicted mirtrons depended on splicing of the intron, however, a number of them were still produced when splicing was abolished by mutation of the splice sites. Surprisingly, the abundance of these same splicing-independent mirtrons was not reduced by knockdown of the microprocessor component, DGCR8. Our results suggest that there may be a subclass of microRNAs that can be processed from a primary RNA transcript by a currently unknown mechanism that does not involve the traditional mirtron or microprocessor pathways.
Keywords: Mirtron, microRNA biogenesis, Splicing
Abstract:
To determine the relative importance of transcriptional regulation vs. RNA processing and turnover during the transition from proliferation to meiotic differentiation in the fission yeast Schizosaccharomyces pombe, we analyzed temporal profiles and effects of RNA surveillance factor mutants on expression of 32 meiotic genes. Comparing nascent transcription with steady state RNA accumulation reveals that the vast majority of these genes show a lag between maximal RNA synthesis and peak RNA accumulation. During meiosis, total RNA levels parallel 3' processing, which occurs in multiple temporally distinct waves that peak from 3-6 hours after meiotic induction. Most early genes and one middle gene, mei4, share a regulatory mechanism in which a specialized RNA surveillance factor targets newly synthesized transcripts for destruction. Mei4p, a member of the forkhead transcription factor family, in turn regulates a host of downstream genes. Remarkably, a spike in transcription is observed for less than one-third of the genes surveyed, and even these show evidence of RNA-level regulation. In aggregate, our findings lead us to propose that a regulatory cascade driven by changes in processing and stability of newly synthesized transcripts operates alongside the well-known transcriptional cascade as fission yeast cells enter meiosis.
Keywords: transcription, polyadenylation, meiosis
Abstract:
Ribosome biogenesis is a complex, dynamic process that must coordinate processing and folding of rRNA precursors with binding of r-proteins. At least 180 assembly factors are required for this process. To understand the mechanism of ribosome assembly, we are studying one particular step- exonucleolytic processing of 27SA3 pre-rRNA to form 27SBS pre-rRNA, and generate the 5´ end of 5.8S rRNA. Depletion of seven assembly factors, collectively referred to as the A3 factors, specifically results in accumulation of 27SA3 pre-rRNA and reduced levels of 27SBS pre-rRNA, suggesting that they function in processing 27SA3 pre-rRNA. Three 5´-3´ exonucleases, Rat1, Xrn1, and Rrp17, are required for this processing step. Six A3 factors associate with preribosomes in an interdependent manner, and are required for the stable association of Rrp17, but not Rat1 or Xrn1 with preribosomes. In the absence of these A3 factors, four r-proteins- rpL17, rpL26, rpL35, and rpL37 cannot associate with preribosomes. These four r-proteins are present close to each other in mature ribosomes; rpL17 and rpL26 bind helix 2 formed by base-pairing between the 5´ end of 5.8S rRNA and 25S rRNA. Further investigation of rpL17 revealed that it becomes stably associated with 66S preribosomes after 27SA3 pre-rRNA processing. In the absence of rpL17, Rat1 cannot stop at the 5´ end of 5.8S rRNA, suggesting that rpL17 serves as the roadblock to Rat1 during 27SA3 pre-rRNA processing. In A3 factor mutants, where rpL17 and the other three r-proteins are absent, Rat1 functions as a surveillance tool for turnover of aberrant assembly intermediates. We propose that A3 factors play a role in bringing together 5.8S and 25S rRNAs to enable formation of helix 2. Binding of rpL17 enabled by A3 factors, stabilizes this helix and ensures that Rat1 stops at the correct site to generate the 5´ end of 5.8S rRNA. When ribosome assembly is perturbed, Rat1 functions as a quality control system to eliminate defective pre-rRNAs.
Keywords: ribosome assembly, pre-rRNA processing, exonucleases
Abstract:
The accumulation of unfolded proteins in the endoplasmic reticulum triggers a stress response program that protects the cells early in the unfolded protein response (UPR) and can lead to apoptosis during prolonged stress. Upon the UPR, ER associated signaling pathways are activated. Early in the stress response, activation of the ER trans-membrane protein PERK causes increased phosphorylation of eIF2α, resulting in a global decrease of protein synthesis and increased synthesis of the transcription factor ATF4 which promotes expression of stress response genes. Later in the response, dephosphorylation of eIF2α allows translation of the transcribed stress response mRNAs. Early in the response, activation of the ER-transmembrane protein, IRE1α causes cytoplasmic splicing of a 26 nt intron from the ER-associated unspliced XBP1 mRNA. This splicing generates the active XBP1 transcription factor that induces the expression of genes involved in protein folding and degradation. The current view is that these two pathways are activated independently during the UPR. We show here that phosphorylation of eIF2α is required for maximal induction of the active XBP1 levels via a mechanism that involves stabilization of the spliced XBP1 mRNA. Using cells deficient for eIF2α phosphorylation, we show that splicing of the XBP1 mRNA is independent of eIF2α phosphorylation, but enhanced stabilization of the spliced mRNA requires global inhibition of protein synthesis (caused by eIF2α phosphorylation) which induces disassembly of ribosomes. We propose a model where early in the ER stress response, the unspliced XBP1 mRNA is spliced, generating a translationally repressed mRNA at a time when global protein synthesis is inhibited. Stabilization of the spliced mRNA allows its accumulation followed by translation during the subsequent translational recovery. We conclude that eIF2α-phosphorylation-mediated control of mRNA turnover amplifies the signals that promote the ER’s capacity to handle protein folding during stress.
Keywords: XBP1, mRNA stability
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
Cyclic diguanylate (c-di-GMP) is a second messenger signaling molecule important for the regulation of many bacterial processes, including virulence, motility, and biofilm formation. Recent developments have shown that c-di-GMP is recognized by a riboswitch, an mRNA domain that controls gene expression in response to varying concentrations of a target ligand. The c-di-GMP riboswitch has been shown to undergo a global structural rearrangement in response to ligand binding. We have used single molecule fluorescence resonance energy transfer (smFRET) to investigate this conformational change. In the absence of c-di-GMP, the riboswitch exists in primarily an undocked conformation and only in the presence of the correct substrate can it fold into a stable docked conformation. This docked conformation was found to be stable for at least 30 minutes. As this riboswitch is responsible for the regulation of many critical bacterial processes, the stability of the docked conformation in the presence of c-di-GMP may be important to ensure a constant expression of required genes. In the presence of c-di-AMP, the riboswitch displayed conformational behavior similar to that in the absence of substrate, demonstrating the specificity of this riboswitch. A population of dynamic molecules that exists primarily in a folded conformation was found to increase in the presence of high magnesium concentrations leading to the idea that magnesium ions aid in the conformational switch from an undocked to folded conformation but are not able to fully stabilize the docked conformation.
Keywords: single molecule FRET, riboswitch, c-di-GMP
Abstract:
Conformational changes are important in RNA for both binding and catalysis. A conformational change of a non-canonical pair was modeled computationally. In an NMR structure of an AA mismatch in the sequence:
5’ GGUGAAGGCU3’
3’PCCGAAGCCG 5’
where P is Purine, the AA non-canonical pair was in conformational exchange between a minor and major conformation1. The conversion of the trans Hoogsteen-sugar to a different trans sugar-Hoogsteen non-canonical pair occurs on the order of tens of microseconds.
The AMBER molecular mechanics software package2 was used to model conformational change pathways. Nudged Elastic Band (NEB)3 was used to predict minimal potential energy paths for a series of all-atom images of the system along the path. Predicted pathways from NEB2 were analyzed and a reaction coordinate determined for the conformational change. Umbrella sampling4 was then used to predict the free energy profile5. Umbrella sampling was done using 25 windows of 10 degrees each with 12 ns of sampling per window for 6 different random number seeds. Total sampling involved 1.8 microseconds of MD spanning about 2 years of CPU time. Additionally, the Molecular Mechanics Poisson Boltzmann and Generalized Born Surface Area (MM-PBSA/GBSA) methods were also used to estimate the free energy change between conformations. The opposite relative stability of two structures was predicted, which suggests improvements can be made in the AMBER force field.
References:
1. Chen, et al. 2006. An alternating sheared AA pair and elements of stability for a single
sheared purine-purine flanked by sheared GA pairs in RNA. Biochem. 45:6889-6903
2. Case, et al. 2005. The AMBER Biomolecular Simulation Programs. J. Comp. Chem. 26: 1668-1688
3. Mathews & Case. 2006. Nudged elastic band calculation of minimal energy paths for the
conformational change of a GG non-canonical pair. JMB. 357:1683-1593
4. Torrie & Valleau. 1977. Nonphysical sampling distributions in Monte Carlo free-energy estimation: Umbrella sampling. J. Comp. Phys. 23:187-199
5. Kumar, et al. 1992. The Weighted Histogram Analysis Method for Free Energy Calculations of Biomolecules. J. Comp. Chem. 1992. 13:1011-1021
Keywords: Conformational Change, Molecular Mechanics, Free Energy
Abstract:
Riboswitches regulate expression of bacterial genes through the formation of specific secondary structures in their 5' UTRs. We employ kinetic Monte Carlo simulation to model the structure formation during transcription of transcriptional terminators and Shine-Dalgarno sequesterers. Transcriptional terminators fold with higher efficiency than Shine-Dalgarno sequesterers. We suggest this difference is the result of natural selection for efficient folding of terminator hairpins which must fold quickly and reliably in order to successfully terminate transcription. In contrast, Shine-Dalgarno sequesterers act at the time of translation, reducing the selective pressure for high folding efficiency.
Keywords: Riboswitch, termination, Folding kinetics
Abstract not available online - please check the printed booklet.
Abstract:
Catalytic RNAs, or ribozymes, starkly demonstrate the ability of 'naked' RNA to adopt complex three-dimensional architectures that position chemical groups with high precision. Ribozymes play fundamental roles in biological processes such as the homeostatic control of gene expression, genomic replication and protein synthesis. In this lecture, I will discuss studies of catalytic RNAs that emphasize two main experimental approaches. By chemically capturing ribozymes in different functional states and determining their three-dimensional structures we have generated crystallographic 'movies' of these catalysts in action. These studies have produced insights into the molecular basis of ligand and coenzyme recognition and catalysis. We have also employed a combination of in vitro evolution and next generation sequencing to study the fitness landscape of ribozymes. By analyzing the phenotypic fitness of tens of millions of genotypes, these studies reveal the functional importance of each nucleotide, and complement biochemical and structural studies, which are typically restricted to a wild-type sequence and a handful of mutants.
Keywords: Ribozymes, Crystallography, In vitro evolution
Abstract not available online - please check the printed booklet.
Abstract:
The flagellate protozoan T. brucei causes an invariably fatal disease, Human African Trypanosomiasis (HAT) or sleeping sickness. Unfortunately, there is no anti-HAT vaccine, yet available drugs are few, highly toxic, and have inadequate efficacy. New, superior drugs are needed, necessitating identification and characterization of novel drug targets. Recently, RNA-binding pentatricopeptide repeat (PPR) proteins were identified to be expanded in number and highly conserved among the trypanosomatids, and hence are potential drug targets. We have embarked on understanding the structure and RNA-binding activity of a 26.9 kDa PPR protein (here called PPR27), the smallest member among at least 28 T. brucei PPR proteins. By refolding of protein from thioredoxin-PPR27 fusion protein inclusion bodies, we were able to determine the regular conformation and oligomeric state of PPR27, as well as obtain preliminary insights into the RNA sequence selectivity of PPR27. We have built on this and expressed soluble PPR27 as a C-terminal fusion to maltose binding protein (MBP). We have generated a panel of N-terminal and C-terminal deletion mutants of PPR27 with the object of elucidating the contribution of the various PPR motifs to RNA binding.
Keywords: T brucei, pentatricopeptide repeat, protein
Abstract:
More than 80% of the population is infected by herpes simplex virus 1 (HSV-1). It is known for causing cold sores and infects mucous membranes, moreover, it can cause severe infections to newborns and immunosuppressed individuals. HSV-1 is an enveloped virus. It has a double stranded DNA genome that codes for more than 80 genes, thus necessitating effective genetic regulation. Viral genes are expressed in a temporal cascade of immediate-early, early and late kinetics. One of the modes of regulation utilized by HSV-1 is alternative polyadenylation: a post-transcriptional event that allows one gene to use two different polyadenylation signals. This process generates transcripts that differ in their 3’ untranslated region (3’UTR), which can affect translation efficiency among other things. One of the HSV-1 genes whose expression is regulated by alternative polyadenylation is UL24. Short UL24 transcripts arise from use of the UL24 polyadenylation signal and are expressed with early kinetics. Long, polycistronic UL24 transcripts arise from use of the UL26 polyadenylation signal, and these transcripts exhibit late kinetics. We hypothesized that the switch from short to long UL24 transcripts is due to changes in levels of polyadenylation factors over the course of infection. To test this hypothesis, we prepared lysates from Vero and HeLa cells at early and late times following infection. Levels of various cellular polyadenylation factors were assessed by Western blotting. We are also using an RNA affinity approach to isolate proteins that interact with the 3’UTRs of the long and short UL24 transcripts. The results from this project will contribute to a better understanding of post-trancriptional regulation during HSV-1 infection.
Keywords: gene regulation, RNA binding proteins, viral gene expression
Abstract:
Many transposable elements and viruses rely on tRNAs and tRNA-like structures during their life cycles. To initiate reverse transcription, HIV-1 anneals human tRNALys3 to the complementary primer binding site (PBS), and cDNA synthesis begins from the 3´-OH of the tRNA. We have previously shown that human lysyl-tRNA synthetase (LysRS) is specifically incorporated into HIV-1 by the Gag protein and that this interaction is critical for tRNALys3 packaging. Herein, we describe a novel conserved tRNALys3-like element (TLE) in the 5´-UTR of the HIV-1 genome proximal to the PBS. Using electrophoretic mobility shift assays and fluorescence anisotropy binding studies, we show that LysRS interacts with the TLE, which mimics the anticodon domain of tRNALys3, whereas human TrpRS and ProRS do not. The affinity of LysRS for short (23 nt) HIV-1 genome-derived RNA hairpins containing the TLE is similar to that of in vitro transcribed 76-nt tRNALys3 (Kd ~ 90 nM). Human LysRS recognizes tRNALys isoacceptors via a specific interaction with the anticodon (UUU in tRNALys3 and CUU in tRNALys1,2). Mutation of the UUU motif in the TLE to AAA or CCC greatly reduces the LysRS-TLE interaction (Kd ≥ 2.5 and 8 uM, respectively). Longer 330-nt RNAs derived from the 5´-UTR compete even more effectively for LysRS binding to a tRNALys3 anticodon stem-loop mimic than tRNALys3, suggesting that in addition to the U-rich motif in the TLE, other sequence elements in the 5´ UTR contribute to preferential binding of LysRS to the HIV-1 genome. 293T cells transfected with virus containing TLE mutations show no change in the level of packaged tRNA but result in a 70% decrease in the amount of tRNALys3 placed onto the genome. These new findings suggest that the HIV-1 genome uses molecular mimicry of tRNALys3 to facilitate primer release from LysRS and placement onto the PBS.
Keywords: tRNA-like element, HIV genome, reverse transcription
Abstract not available online - please check the printed booklet.
Abstract:
In eukaryotes ribosome assembly requires hundreds of conserved essential proteins not present in the mature particle. Despite their importance, their function remains unknown. This is oftein because protein deletion affects the composition of the entire particle. Furthermore, many proteins appear to be present in assembling ribosomes for extended times making it difficult to pinpoint their role to a particular step. Here we have combined classical yeast biochemistry with experiments using recombinant proteins and RNA to study the role of Dim2, and its interaction with Nob1, the nuclease that generates the 3’-end of 18S rRNA. Analysis of Dim2 mutants in which the interaction with Nob1 is disrupted demonstrates that the interaction between Dim2 and Nob1 is essential. Furthermore, our data also indicate that Dim2 helps position Nob1 for correct cleavage at the 3’-end of 18S rRNA, as a point mutation that abolishes this interaction in vitro leads to accumulation of pre-ribosomes containing Nob1 and 20S rRNA. Interestingly, the site of interaction with Nob1 is mapped to a KH-like domain in Dim2, suggesting that this domain typically used for RNA binding can also be used as a protein-recognition motif.
Keywords: Ribosome Assembly, Dim2, KH domain
Abstract:
Prolyl-tRNA synthetases (ProRSs) mischarge cognate tRNAPro with the smaller noncognate amino acid Ala, and with Cys, which is of similar size as cognate Pro. Bacterial ProRSs possess a discrete editing active site (INS domain) that functions to selectively hydrolyze mischarged Ala- but fails to hydrolyze Cys-tRNAPro, which is edited by a free-standing domain, YbaK, via substrate sulfhydryl side chain chemistry. The molecular basis of substrate specificity of these homologous editing domains is further investigated here. Computational docking studies of E. coli (Ec) ProRS INS bound to CCA-Ala revealed that the substrate Ala binds in a small hydrophobic pocket defined by conserved residues I263, L266 and T277. This pocket is large enough to accommodate Ser- and 2-aminobutyric acid (Abu)-tRNAPro, but cannot hydrolyze Cys- and Pro-tRNAPro. Deacylation activities of INS mutants (I263A, L266A, T277S and I263A/L266A) were tested against Ala-, Pro-, Cys-, Ser- and 2Abu-tRNA substrates. All mutants showed a significant loss in Ala- and 2-Abu deacylation activity and interestingly, I263A gained Cys-deacylation specificity. The G30 residue in YbaK, which aligns with I263, was substituted with Ile to mimic the binding pocket of the INS domain. G30I YbaK failed to hydrolyze either Ala- or Cys-tRNAPro. Thus, restricting the binding pocket size of YbaK prevents Cys-tRNA editing, as expected, but does not allow hydrolysis of Ala. QM/MM calculations suggest that hydrolysis by INS is mediated by a nucleophilic water molecule activated by the 2ʹOH of A76 of tRNAPro. In support of this idea, 2ʹ-deoxy-A76-tRNAPro was severely reduced in INS Ala-deacylation activity. In conclusion, the specificity of substrate-assisted water-mediated hydrolysis by INS is largely dictated by binding pocket size. In contrast, tuning the binding pocket size of YbaK is not sufficient to alter substrate specificity, providing further support for the role of the side chain sulfhydryl in cleavage chemistry.
Keywords: Prolyl-tRNA Synthetase, YbaK, Substrate Specificity, Computational Docking, QM-MM
Abstract:
In splicing, exons are joined together and introns removed to generate the correct RNA for translation. The byproduct of splicing is the removed lariat intron. Processing of the lariat intron is performed by debranching enzyme Dbr1, a 2'-5' phosphodiesterase that is well conserved among all eukaryotes. Dbr1 may also have alternative cellular functions, as S. cerevisiae Dbr1 mutants are defective in Ty1 retrotransposition. However, the function of Dbr1 in retrotransposition is unclear. Better systems are needed to characterize Dbr1 and gain more information about the structure and specificity of the enzyme. Previous reports on Dbr1 characterization use non-natural substrates – as the methods of synthesizing the RNA limited the sequence so that the substrates were not the consensus sequences of lariat RNA. Recently, we have developed the solid-phase synthesis of branched RNA that is a mimic of the lariat RNA - including the branched RNA with conserved sequences as well as unnatural branched RNA and DNA-RNA chimeric analogues. The cleavage and binding analyses of these branched RNAs and branched RNA-DNA chimera with Dbr1 offer insight into the substrate specificity of Dbr1. The use of these analogues and Dbr1 and mutants are being investigated for use in protein-RNA crystallization to further our understanding of debranching of RNA and its role in cellular processes.
Keywords: debranching enzyme, branched RNA, splicing
Abstract:
Usher syndrome is the leading genetic cause of combined blindness and deafness. Until recently, an obstacle to developing treatment strategies for the disease has been the lack of animal models that develop both auditory and visual defects. One mouse model for Usher syndrome that exhibits both phenotypes is based on a mutation in the USH1C gene, USH1C/G216A. This mutation causes the activation of a cryptic 5’ splice site that is used preferentially over the normal site. Splicing from the cryptic site produces a truncated mRNA and protein product. This mouse model provides a valuable tool to investigate therapeutic strategies for Usher syndrome. We used antisense oligonucleotides (ASO) targeted to the cryptic 5’ splice site to correct splicing of Ush1C pre-mRNA and restore proper USH1C expression. ASOs were first tested in a cell-free splicing assay and further validated using an USH1C/G216A minigene expression system. Optimized ASOs were then tested on the endogenous USH2C/G216A transcript and were found to completely correct splicing in cell lines derived from Usher syndrome patients as well as in cell lines derived from USH1C/G216A mouse tissues. The most effective ASO also corrected splicing in multiple tissues when injected into USH1C/G216A mice. Our results suggest therapeutic potential of ASOs in Usher syndrome and other diseases caused by mutations that disrupt splicing.
Keywords: Usher syndrome, Splicing, RNA and disease
Abstract:
Alternative splicing of the HIV-1 genome is necessary for translation of the complete viral proteome. Host proteins, such as hnRNP A1, are used to regulate splicing at the various donor and acceptor sites along the genome. One such site regulated by hnRNP A1 is the conserved 3’ acceptor splice site A7 (ssA7). Silencing of splicing at this site is necessary in order to retain the Rev Responsive Element (RRE) in the adjacent tat/rev intron. The RRE is responsible for nuclear export of unspliced and partially spliced transcripts. Previous studies done on the binding of hnRNP A1 to ssA7 have resulted in two disparate mechanisms.
Our research seeks to clarify the binding determinant of hnRNP A1 on ssA7 by developing a model that will correlate ssA7 and hnRNP A1 structure to their splicing function. The 3D structure of the RNA composing the splice site is being examined by NMR while ITC is being used to study the thermodynamic model of hnRNP A1/ssA7 binding. NMR is also being used to examine which residues of hnRNP A1 are involved in the binding with ssA7.
The ssA7 structure contains three stem loops (SL1, SL2, and SL3). NMR has been used to determine the 3D structure of the third stem loop. For the ITC experiments, the hnRNP A1 analog UP1 was used. UP1 is composed of the two RNA-recognition motifs of hnRNP A1 and is missing the C-terminus, which is responsible for hnRNP A1 self-association. Preliminary ITC experiments examining the binding of UP1 along ssA7 showed that UP1 was able to assemble cooperatively along ssA7 without any self-association. A further truncated form of hnRNP A1, containing just the first RNA-recognition motif, was used in the NMR studies examining binding with SL3 of ssA7. Specific chemical shift changes were observed in the spectrum for residues of RRM1 involved in the binding with RNA. The chemical shift changes map to one surface of RRM1, containing the four stranded beta-pleated sheet and other elements of secondary structure.
References:
Saliou, J., Bourgeois, C., Avadi-Ben Mena, L., Ropers D, Jacquenet S, Marchand V, Stevenin J, Branlant C. Role of RNA structure and protein factors in the control of HIV-1 splicing. Front. Biosci. (2009) 14:2714-2729.
Mayeda, A,, Munroe, S., Caceres, J., and Krainer, A. Function of conserved domains of hnRNP A1 and other hnRNP A/B proteins. EMBO J. (1994) 13:5483-5495.
Marchand V, Méreau A, Jacquenet S, Thomas D, Mougin A, Gattoni R, Stévenin J, Branlant C. A Janus splicing regulatory element modulates HIV-1 tat and rev mRNA production by coordination of hnRNP A1 cooperative binding. J. Mol. Biol. (2002) 323:629-652.
Ding, J., Hayashi, M.K., Zhang, Y. Crystal Structure of the two-RRM domain of hnRNP A1 (UP1) complexed with single-stranded telomeric DNA. Gene Dev. (1999) 13:1102-1115.
Keywords: HIV, Splicing, NMR
Abstract not available online - please check the printed booklet.
Abstract:
The increasing interest in RNA nanotechnology and the demonstrated feasibility of using RNA nanoparticles as therapeutics have prompted the need for imaging systems with nanometer-scale resolution for RNA studies. Phi29 dimeric pRNAs can serve as building blocks in assembly into the hexameric ring of the nanomotors, as modules of RNA nanoparciles, and as vehicles for specific delivery of therapeutics to cancers or viral infected cells. The understanding of the 3D structure of this novel RNA dimeric particle is fundamentally and practically important. Although a 3D model of pRNA dimer has been proposed based on biochemical analysis, no distance measurements or X-ray diffraction data have been reported. Here we evaluated the application of our customized single-molecule dual-viewing system for distance measurement within pRNA dimers using single-molecule Fluorescence Resonance Energy Transfer (smFRET). Ten pRNA monomers labeled with single donor or acceptor fluorophores at various locations were constructed and eight dimers were assembled. smFRET signals were detected for six dimers. The tethered arm sizes of the fluorophores were estimated empirically from dual-labeled RNA/DNA standards. The distances between donor and acceptor were calculated and used as distance parameters to assess and refine the previously reported 3D model of the pRNA dimer. Distances between nucleotides in pRNA dimers were found to be different from those of the dimers bound to procapsid, suggesting a conformational change of the pRNA dimer upon binding to the procapsid.
References:
Dual-channel single-molecule fluorescent resonance energy transfer to establish distance constraints for RNA nanoparticles, Shu D, Zhang H, Petrenko R, Meller J and Guo P, ACS Nano, accepted
Keywords: RNA 3D structure, DNA packaging motor, nanotechnolgoy
Abstract:
The probing and characterization of the behavior of individual nucleic acid based molecular robots is presented, using real-time single-particle tracking with super-resolution total internal reflection fluorescence microscopy (TIRFM). Nucleic acid based molecular assemblies, called “spiders”, implemented as robots with multiple deoxyribozyme sensor-actuator legs traverse and cleave pseudo-one- and two-dimensional landscapes of surface bound chimeric oligonucleotide substrates. We analyze the movement of spiders to determine the characteristics of their random and directional walks on various substrates and demonstrate their ability to respond to specific cues programmed onto the surface landscape. The experimental approach demonstrated here should allow for control over the cybernetic properties of spiders, resulting in the integration and synthesis of complex robotic behaviors at the nanoscale based on RNA and DNA nanotechnology.
Keywords: nucleic acid, computation, motors
Abstract:
The hepatitis delta virus (HDV) ribozyme and HDV-like ribozymes are self-cleaving RNAs found throughout all kingdoms of life. These RNAs fold into a double-nested pseudoknot structure and cleave RNA, yielding 2',3'-cyclic phosphate and 5'-hydroxyl termini. The active site nucleotide C75 has a pKa shifted >2 pH units toward neutrality and has been implicated as a general acid/base in the cleavage reaction. An active site Mg2+ ion that helps activate the 2'-hydroxyl for nucleophilic attack has been characterized biochemically; however, this ion has not been visualized in any previous structures. To create a snapshot of the ribozyme in a state poised for catalysis, we have crystallized and determined the structure of the HDV ribozyme bound to an inhibitor RNA containing a deoxynucleotide at the cleavage site. This structure includes the wild-type C75 nucleotide and Mg2+ ions, both of which are required for maximal ribozyme activity. This structure suggests that the position of C75 does not change during the cleavage reaction. A partially hydrated Mg2+ ion is also found within the active site where it interacts with a newly resolved G•U reverse wobble. Although the inhibitor exhibits crystallographic disorder, we modeled the ribozyme-substrate complex using the conformation of the inhibitor strand observed in the hammerhead ribozyme. This model suggests that the pro-RP oxygen of the scissile phosphate and the 2'-hydroxyl nucleophile are innersphere ligands to the active site Mg2+ ion. Thus, the HDV ribozyme may use a combination of metal ion Lewis acid and nucleobase general acid strategies to effect RNA cleavage.
Keywords: ribozyme, mechanism, HDV
Abstract:
We have measured the rates of transcription and splicing of genes in their natural in vivo chromatin environment. We used DRB to reversibly block new rounds of transcription in cultured cells. During DRB treatment, elongating RNA polymerases exited the genes and pre-mRNA was processed. Upon release from DRB, new rounds of transcription began within minutes and could be monitored using qRT-PCR of pre-mRNAs. Using this method, we found that transcription elongation proceeded at a rate of about 3.8 kb/min and that splicing occurred co-transcriptionally within 5-10 minutes of synthesis of the downstream exon. Remarkably, this splicing rate was independent of intron length between 1 kb and 250 kb. We have further shown that these results are essentially the same for short (30 min) and long (3 hr) DRB treatments as well as when flavopiridol is used in place of DRB. We also see similar results using interferon induction of genes instead of DRB treatment (1).
We have also used this method to follow the kinetics of transcription and splicing of long introns. In Drosophila, long introns are spliced in multiple steps by a recursive mechanism in a 5’ to 3’ order. It has been unclear whether long mammalian introns are spliced via a single event or multiple events. When we analyzed a 107 kb human intron, we found that transcription proceeded uniformly through the intron. Multiple sites within the intron were monitored for the kinetics of appearance and disappearance of intronic RNA. We found that all intronic RNA signals increased from the time of synthesis until 5 minutes after synthesis of the downstream exon and appearance of spliced RNA. The signal at the 5’ exon/intron junction was stable until splicing occurred at the normal 3’ splice site showing that recursive splicing did not occur. No evidence was seen for internal splicing events within the intron. This data strongly suggests that this intron is spliced out in a single event. Remarkably, the entire 107 kb intron appeared to be stable until shortly after splicing at which point all the intronic signals decreased in parallel consistent with rapid degradation.
References:
1. Singh, J. and Padgett, R.A. (2009) Rates of in situ transcription and splicing of large human genes. Nat. Struct. Molec. Biol. 16: 1128-1133.
Keywords: Splicing, Transcription, Chromatin
Abstract not available online - please check the printed booklet.
Abstract:
U6 and U2 spliceosomal small nuclear RNAs (snRNAs) play central roles in splicing, an ubiquitous and essential step in eukaryotic gene expression. In activated spliceosomes these two snRNAs form a functionally-critical basepaired complex and evidence indicates that in vitro-assembled complexes of these two snRNAs can perform an in vitro splicing reaction9[1]. However, despite its functional importance, structural information on the U6/U2 basepaired complex has been lacking. Here we describe small-angle X-Ray scattering (SAXS) structural analyses of an in vitro-assembled, base-paired complex formed by central domains of human U6 and U2 snRNAs. The results indicate the snRNAs fold into a four-way junction-like structure [2], in which the intramolecular stemloop of U6 and the ACAGAGA-containing stem are co-axially stacked, with the ISL and helix II showing side-by-side stacking. Our data suggests that the spontaneous folding of the snRNAs is modified by spliceosomal proteins, which help juxtapose functionally-critical residues, and thus indicates a RNA chaperone role for proteins in the spliceosomal active site.
References:
[1] Valadkhan, S.; Mohammadi, A.; Jaladat, Y.; Geisler, S., 2009, Protein-free small nuclear RNAs catalyze a two-step splicing reaction, Proc. Natl Acad Sci USA, 106(29), 11901-11906.
[2] Lilley, D. M. J., Quart. Rev. Biophys. 2000. 33, 109-159.
Keywords: model spliceosome, Synchrotron SAXS, four-way junction
