Showing papers in "RNA in 2004"
TL;DR: A program is presented, RNA-hybrid, that predicts multiple potential binding sites of miRNAs in large target RNAs and applied this method to the prediction of Drosophila miRNA targets in 3'UTRs and coding sequence.
Abstract: MicroRNAs (miRNAs) are short RNAs that post-transcriptionally regulate the expression of target genes by binding to the target mRNAs. Although a large number of animal miRNAs has been defined, only a few targets are known. In contrast to plant miRNAs, which usually bind nearly perfectly to their targets, animal miRNAs bind less tightly, with a few nucleotides being unbound, thus producing more complex secondary structures of miRNA/target duplexes. Here, we present a program, RNA-hybrid, that predicts multiple potential binding sites of miRNAs in large target RNAs. In general, the program finds the energetically most favorable hybridization sites of a small RNA in a large RNA. Intramolecular hybridizations, that is, base pairings between target nucleotides or between miRNA nucleotides are not allowed. For large targets, the time complexity of the algorithm is linear in the target length, allowing many long targets to be searched in a short time. Statistical significance of predicted targets is assessed with an extreme value statistics of length normalized minimum free energies, a Poisson approximation of multiple binding sites, and the calculation of effective numbers of orthologous targets in comparative studies of multiple organisms. We applied our method to the prediction of Drosophila miRNA targets in 3′UTRs and coding sequence. RNAhybrid, with its accompanying programs RNAcalibrate and RNAeffective, is available for download and as a Web tool on the Bielefeld Bioinformatics Server (http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/).
2,236 citations
TL;DR: Together, these data show that human pri-miRNAs are not only structurally similar to mRNAs but can, in fact, function both as pri- miRNAs and m RNAs.
Abstract: The factors regulating the expression of microRNAs (miRNAs), a ubiquitous family of approximately 22-nt noncoding regulatory RNAs, remain undefined. However, it is known that miRNAs are first transcribed as a largely unstructured precursor, termed a primary miRNA (pri-miRNA), which is sequentially processed in the nucleus, to give the approximately 65-nt pre-miRNA hairpin intermediate, and then in the cytoplasm, to give the mature miRNA. Here we have sought to identify the RNA polymerase responsible for miRNA transcription and to define the structure of a full-length human miRNA. We show that the pri-miRNA precursors for nine human miRNAs are both capped and polyadenylated and report the sequence of the full-length, approximately 3433-nt pri-miR-21 RNA. This pri-miR-21 gene sequence is flanked 5' by a promoter element able to transcribe heterologous mRNAs and 3' by a consensus polyadenylation sequence. Nuclear processing of pri-miRNAs was found to be efficient, thus largely preventing the nuclear export of full-length pri-miRNAs. Nevertheless, an intact miRNA stem-loop precursor located in the 3' UTR of a protein coding gene only moderately inhibited expression of the linked open reading frame, probably because the 3' truncated mRNA could still be exported and expressed. Together, these data show that human pri-miRNAs are not only structurally similar to mRNAs but can, in fact, function both as pri-miRNAs and mRNAs.
1,916 citations
TL;DR: Nuclear export of pre-miRNAs is studied and it is shown that the process is saturable and thus carrier-mediated, and that exportin 5 interacts with double-stranded RNA in a sequence-independent manner.
Abstract: microRNAs (miRNAs) are widespread among eukaryotes, and studies in several systems have revealed that miRNAs can regulate expression of specific genes Primary miRNA transcripts are initially processed to approximately 70-nucleotide (nt) stem-loop structures (pre-miRNAs), exported to the cytoplasm, further processed to yield approximately 22-nt dsRNAs, and finally incorporated into ribonucleoprotein particles, which are thought to be the active species Here we study nuclear export of pre-miRNAs and show that the process is saturable and thus carrier-mediated Export is sensitive to depletion of nuclear RanGTP and, according to this criterion, mediated by a RanGTP-dependent exportin An unbiased affinity chromatography approach with immobilized pre-miRNAs identified exportin 5 as the pre-miRNA-specific export carrier We have cloned exportin 5 from Xenopus and demonstrate that antibodies raised against the Xenopus receptor specifically block pre-miRNA export from nuclei of Xenopus oocytes We further show that exportin 5 interacts with double-stranded RNA in a sequence-independent manner
1,394 citations
TL;DR: It is shown that 2'-O-methyl oligoribonucleotides, but not 2'-deoxyoligon nucleotides specifically inactivate the RNAi activity associated with miRNA-protein complexes in human cell extracts as well as in cultured human cells.
Abstract: A large number of miRNAs have recently been discovered in plants and animals. Development of reverse genetic approaches that act to inhibit microRNA function would facilitate the study of this new class of noncoding RNA. Here we show that 2'-O-methyl oligoribonucleotides, but not 2'-deoxyoligonucleotides specifically inactivate the RNAi activity associated with miRNA-protein complexes in human cell extracts as well as in cultured human cells.
752 citations
TL;DR: It is demonstrated that siRNA modified with 2'-fluoro (2'-F) pyrimidines are functional in cell culture and have a greatly increased stability and a prolonged half-life in human plasma as compared to 2'-OH containing siRNAs.
Abstract: Chemical modifications have been incorporated into short interfering RNAs (siRNAs) without reducing their ability to inhibit gene expression in mammalian cells grown in vitro. In this study, we begin to assess the potential utility of 2-modified siRNAs in mammals. We demonstrate that siRNA modified with 2-flouro (2-F) pyrimidines are functional in cell culture and have a greatly increased stability and a prolonged half-life in human plasma as compared to 2-OH containing siRNAs. Moreover, we show that the 2-F containing siRNAs are functional in mice and can inhibit the expression of a target gene in vivo. However, even though the modified siRNAs have greatly increased resistance to nuclease degradation in plasma, this increase in stability did not translate into enhanced or prolonged inhibitory activity of target gene reduction in mice following tail vein injection. Thus, this study shows that 2-F modified siRNAs are functional in vivo, but that they are not necessarily more potent than unmodified siRNAs in animals.
580 citations
TL;DR: expression profiling in mammalian tissue-culture cells treated under standard conditions with conventional 21-bp siRNAs is performed and finds, unexpectedly, that >1000 genes involved in diverse cellular functions are nonspecifically stimulated or repressed.
Abstract: RNA interference is an evolutionarily conserved process in which expression of a specific gene is post-transcriptionally inhibited by a small interfering RNA (siRNA), which recognizes a complementary mRNA and induces its degradation. Currently, RNA interference is being used extensively to inhibit expression of specific genes for experimental and therapeutic purposes. For applications in mammalian cells, siRNAs are designed to be 1000 genes involved in diverse cellular functions are nonspecifically stimulated or repressed. The effects on gene expression are dependent upon siRNA concentration and are stable throughout the course of siRNA treatment. Our results can be explained by previous studies showing that dsRNAs can affect multiple signaling and transcription pathways in addition to PKR. The potential for this widespread, nonspecific effect on mammalian gene expression must be carefully considered in the design of siRNA experiments and therapeutic applications.
560 citations
TL;DR: The energetic factors that must be considered when ions interact with two different RNA environments are summarized and an important conclusion is that diffuse ions are a major factor in the stabilization of RNA tertiary structures.
Abstract: RNA folding into stable tertiary structures is remarkably sensitive to the concentrations and types of cations present; an understanding of the physical basis of ion-RNA interactions is therefore a prerequisite for a quantitative accounting of RNA stability. This article summarizes the energetic factors that must be considered when ions interact with two different RNA environments. “Diffuse ions” accumulate near the RNA because of the RNA electrostatic field and remain largely hydrated. A “chelated” ion directly contacts a specific location on the RNA surface and is held in place by electrostatic forces. Energetic costs of ion chelation include displacement of some of the waters of hydration by the RNA surface and repulsion of diffuse ions. Methods are discussed for computing both the free energy of the set of diffuse ions associated with an RNA and the binding free energies of individual chelated ions. Such calculations quantitatively account for the effects of Mg 2+ on RNA stability where
535 citations
TL;DR: The presence of transcription factors in the spliceosome and the existence of proteins with dual activities in splicing and transcription can explain the links between both processes and add a new level of complexity to the regulation of gene expression in eukaryotes.
Abstract: Transcription and pre-mRNA splicing are extremely complex multimolecular processes that involve protein-DNA, protein-RNA, and protein-protein interactions. Splicing occurs in the close vicinity of genes and is frequently cotranscriptional. This is consistent with evidence that both processes are coordinated and, in some cases, functionally coupled. This review focuses on the roles of cis- and trans-acting factors that regulate transcription, on constitutive and alternative splicing. We also discuss possible functions in splicing of the C-terminal domain (CTD) of the RNA polymerase II (pol II) largest subunit, whose participation in other key pre-mRNA processing reactions (capping and cleavage/polyadenylation) is well documented. Recent evidence indicates that transcriptional elongation and splicing can be influenced reciprocally: Elongation rates control alternative splicing and splicing factors can, in turn, modulate pol II elongation. The presence of transcription factors in the spliceosome and the existence of proteins, such as the coactivator PGC-1, with dual activities in splicing and transcription can explain the links between both processes and add a new level of complexity to the regulation of gene expression in eukaryotes.
501 citations
TL;DR: It is demonstrated that effects of miRNAs on translation can be mimicked in human HeLa cells by the miRNA-independent tethering of Ago proteins to the 3'-UTR of a reporter mRNA.
Abstract: MicroRNAs (miRNAs) are ~21-nt-long RNAs involved in regulating development, differentiation, and other processes in eukaryotes. In metazoa, nearly all miRNAs control gene expression by imperfectly base-pairing with the 3′slated region (3′f target mRNAs and repressing protein synthesis by an unknown mechanism. It is also unknown whether miRNA–mRNA duplexes containing mismatches and bulges provide specific features that are recognized by factors mediating the repression. miRNAs form part of ribonucleoprotein complexes, miRNPs, that contain Argonaute (Ago) and other proteins. Here we demonstrate that effects of miRNAs on translation can be mimicked in human HeLa cells by the miRNA-independent tethering of Ago proteins to the 3′ a reporter mRNA. Inhibition of protein synthesis occurred without a change in the reporter mRNA level and was dependent on the number, but not the position, of the hairpins tethering hAgo2 to the 3′hese findings indicate that a primary function of miRNAs is to guide their associated proteins to the mRNA.
413 citations
TL;DR: It is found that the expression of target mRNAs predicted on the basis of sequence complementarity is unrelated to the tissues in which the corresponding miRNA is expressed, suggesting that miRNAs have widespread regulatory roles in adults.
Abstract: MicroRNAs (miRNAs) are short, stable, noncoding RNAs involved in post-transcriptional gene silencing via hybridization to mRNA. Few have been thoroughly characterized in any species. Here, we describe a method to detect miRNAs using micro-arrays, in which the miRNAs are directly hybridized to the array. We used this method to analyze miRNA expression across 17 mouse organs and tissues. More than half of the 78 miRNAs detected were expressed in specific adult tissues, suggesting that miRNAs have widespread regulatory roles in adults. By comparing miRNA levels to mRNA levels determined in a parallel microarray analysis of the same tissues, we found that the expression of target mRNAs predicted on the basis of sequence complementarity is unrelated to the tissues in which the corresponding miRNA is expressed.
391 citations
TL;DR: Surprisingly, it is shown that the association of the RNA-binding protein HuR with its target mRNA, c-fos, as detected by co-immunoprecipitation, results largely from reassociation of molecules subsequent to cell lysis, demonstrating that co-invention does not always recapitulate the in vivo state of ribonucleoprotein complexes.
Abstract: Immuno- and other affinity-purification approaches are commonly used to characterize the composition of ribonucleoprotein complexes. While associations detected by these procedures are often interpreted as reflecting in vivo interactions, it is also possible that they arise from reassociation of molecules after cell lysis. Here we used an experimental approach that allowed us to distinguish between these possibilities. Surprisingly, we show that the association of the RNA-binding protein HuR with its target mRNA, c-fos, as detected by co-immunoprecipitation, results largely from reassociation of molecules subsequent to cell lysis. The existence of such postlysis reassortments thus demonstrates that co-immunoprecipitation does not always recapitulate the in vivo state of ribonucleoprotein complexes.
TL;DR: A partition function calculation for RNA secondary structure is presented that uses a current set of nearest neighbor parameters for conformational free energy at 37 degrees C, including coaxial stacking to demonstrate pairs with high probability that are therefore well determined as compared to base pairs with lower probability of pairing.
Abstract: A partition function calculation for RNA secondary structure is presented that uses a current set of nearest neighbor parameters for conformational free energy at 37 degrees C, including coaxial stacking. For a diverse database of RNA sequences, base pairs in the predicted minimum free energy structure that are predicted by the partition function to have high base pairing probability have a significantly higher positive predictive value for known base pairs. For example, the average positive predictive value, 65.8%, is increased to 91.0% when only base pairs with probability of 0.99 or above are considered. The quality of base pair predictions can also be increased by the addition of experimentally determined constraints, including enzymatic cleavage, flavin mono-nucleotide cleavage, and chemical modification. Predicted secondary structures can be color annotated to demonstrate pairs with high probability that are therefore well determined as compared to base pairs with lower probability of pairing.
TL;DR: It is shown that human and mouse miRNA22 precursor molecules are subject to posttranscriptional modification by A-to-I RNA editing in vivo and the observed editing events are predicted to have significant implications for the biogenesis and function of mi RNA22.
Abstract: Micro RNAs comprise a large family of small, functional RNAs with important roles in the regulation of protein coding genes in animals and plants. Here we show that human and mouse miRNA22 precursor molecules are subject to posttranscriptional modification by A-to-I RNA editing in vivo. The observed editing events are predicted to have significant implications for the biogenesis and function of miRNA22 and might point toward a more general role for RNA editing in the regulation of miRNA gene expression.
TL;DR: Metal ions are particularly abundant in the region surrounding the peptidyl transferase center, where stabilizing cationic tails of ribosomal proteins are notably absent, which may point to the importance of metal ions for the stabilization of specific RNA structures in the evolutionary period prior to the appearance of proteins.
Abstract: Both monovalent cations and magnesium ions are well known to be essential for the folding and stability of large RNA molecules that form complex and compact structures. In the atomic structure of the large ribosomal subunit from Haloarcula marismortui, we have identified 116 magnesium ions and 88 monovalent cations bound principally to rRNA. Although the rRNA structures to which these metal ions bind are highly idiosyncratic, a few common principles have emerged from the identities of the specific functional groups that coordinate them. The nonbridging oxygen of a phosphate group is the most common inner shell ligand of Mg++, and Mg++ ions having one or two such inner shell ligands are very common. Nonbridging phosphate oxygens and the heteroatoms of nucleotide bases are common outer shell ligands for Mg++ ions. Monovalent cations usually interact with nucleotide bases and protein groups, although some interactions with nonbridging phosphate oxygens are found. The most common monovalent cation binding site is the major groove side of G-U wobble pairs. Both divalent and monovalent cations stabilize the tertiary structure of 23S rRNA by mediating interactions between its structural domains. Bound metal ions are particularly abundant in the region surrounding the peptidyl transferase center, where stabilizing cationic tails of ribosomal proteins are notably absent. This may point to the importance of metal ions for the stabilization of specific RNA structures in the evolutionary period prior to the appearance of proteins, and hence many of these metal ion binding sites may be conserved across all phylogenetic kingdoms.
TL;DR: In a human neuronal cell line, the miRNP proteins eIF2C2 (a member of the Argonaute family of proteins), Gemin3, and Gemin4 along with miRNAs cosediment with polyribosomes suggest that miR NP proteins may play important roles in target mRNA recognition and translational repression.
Abstract: MicroRNAs (miRNAs) are small regulatory RNAs that control gene expression by base-pairing with their mRNA targets. miRNAs assemble into ribonucleoprotein complexes termed miRNPs. Animal miRNAs recognize their mRNA targets via partial antisense complementarity and repress mRNA translation at a step after translation initiation. How animal miRNAs recognize their mRNA targets and how they control their translation is unknown. Here we describe that in a human neuronal cell line, the miRNP proteins eIF2C2 (a member of the Argonaute family of proteins), Gemin3, and Gemin4 along with miRNAs cosediment with polyribosomes. Furthermore, we describe a physical association between a let-7b (miRNA)-containing miRNP and its putative human mRNA target in polyribosome-containing fractions. These findings suggest that miRNP proteins may play important roles in target mRNA recognition and translational repression.
TL;DR: It is shown that eIF4A3 associates preferentially with nuclear complexes containing the EJC proteins magoh and Y14, and likely provides a splicing-dependent influence on the translation of mRNAs.
Abstract: The exon junction complex (EJC) is a protein complex that assembles near exon–exon junctions of mRNAs as a result of splicing. EJC proteins play important roles in postsplicing events including mRNA export, cytoplasmic localization, and nonsense-mediated decay. Recent evidence suggests that mRNA translation is also influenced by the splicing history of the transcript. Here we identify eIF4A3, a DEAD-box RNA helicase and a member of the eIF4A family of translation initiation factors, as a novel component of the EJC. We show that eIF4A3 associates preferentially with nuclear complexes containing the EJC proteins magoh and Y14. Furthermore, eIF4A3, but not the highly related eIF4A1 or eIF4A2, preferentially associates with spliced mRNA. In vitro splicing and mapping experiments demonstrate that eIF4A3 binds mRNAs at the position of the EJC. Using monoclonal antibodies, we show that eIF4A3 is found in the nucleus whereas eIF4A1 and eIF4A2 are found in the cytoplasm. Thus, eIF4A3 likely provides a splicing-dependent influence on the translation of mRNAs.
TL;DR: The Invader miRNA assay, which can be performed in unfractionated detergent lysates, uses fluorescence detection in microtiter plates and requires only 2-3 h incubation time, allowing for parallel analysis of multiple samples in high-throughput screening analyses.
Abstract: The short lengths of microRNAs (miRNAs) present a significant challenge for detection and quantitation using conventional methods for RNA analysis. To address this problem, we developed a quantitative, sensitive, and rapid miRNA assay based on our previously described messenger RNA Invader assay. This assay was used successfully in the analysis of several miRNAs, using as little as 50-100 ng of total cellular RNA or as few as 1,000 lysed cells. Its specificity allowed for discrimination between miRNAs differing by a single nucleotide, and between precursor and mature miRNAs. The Invader miRNA assay, which can be performed in unfractionated detergent lysates, uses fluorescence detection in microtiter plates and requires only 2-3 h incubation time, allowing for parallel analysis of multiple samples in high-throughput screening analyses.
TL;DR: A large-scale analysis of intron retention in a set of 21,106 known human genes suggests that a significant fraction of the observed events are not spurious and might reflect biological significance.
Abstract: Alternative splicing is a very frequent phenomenon in the human transcriptome. There are four major types of alternative splicing: exon skipping, alternative 3 splice site, alternative 5 splice site, and intron retention. Here we present a large-scale analysis of intron retention in a set of 21,106 known human genes. We observed that 14.8% of these genes showed evidence of at least one intron retention event. Most of the events are located within the untranslated regions (UTRs) of human transcripts. For those retained introns interrupting the coding region, the GC content, codon usage, and the frequency of stop codons suggest that these sequences are under selection for coding potential. Furthermore, 26% of the introns within the coding region participate in the coding of a protein domain. A comparison with mouse shows that at least 22% of all informative examples of retained introns in human are also present in the mouse transcriptome. We discuss that the data we present suggest that a significant fraction of the observed events is not spurious and might reflect biological significance. The analyses also allowed us to generate a reliable set of intron retention events that can be used for the identification of splicing regulatory elements.
TL;DR: The improved analysis supports the previous assertion that miRNA gene identification is nearing completion in C. elegans with apparently no more than 20 miRNA genes now remaining to be identified.
Abstract: MicroRNAs are approximately 22-nucleotide (nt) RNAs processed from foldback segments of endogenous transcripts. Some are known to play important gene regulatory roles during animal and plant development by pairing to the messages of protein-coding genes to direct the post-transcriptional repression of these messages. Previously, we developed a computational method called MiRscan, which scores features related to the foldbacks, and used this algorithm to identify new miRNA genes in the nematode Caenorhabditis elegans. In the present study, to identify sequences that might be involved in processing or transcriptional regulation of miRNAs, we aligned sequences upstream and downstream of orthologous nematode miRNA foldbacks. These alignments showed a pronounced peak in sequence conservation about 200 bp upstream of the miRNA foldback and revealed a highly significant sequence motif, with consensus CTCCGCCC, that is present upstream of almost all independently transcribed nematode miRNA genes. Scoring the pattern of upstream/downstream conservation, the occurrence of this sequence motif, and orthology of host genes for intronic miRNA candidates, yielded substantial improvements in the accuracy of MiRscan. Nine new C. elegans miRNA gene candidates were validated using a PCR-sequencing protocol. As previously seen for bacterial RNA genes, sequence features outside of the RNA secondary structure can therefore be very useful for the computational identification of eukaryotic noncoding RNA genes. The total number of confidently identified nematode miRNAs now approaches 100. The improved analysis supports our previous assertion that miRNA gene identification is nearing completion in C. elegans with apparently no more than 20 miRNA genes now remaining to be identified.
TL;DR: U1 snRNA base-pairing with positions +6 and -1 is the only functional requirement for mRNA splicing of this 5' splice site, indicating the importance of U1 sn RNA base- Pairing to the exonic portion of the 5'splice site.
Abstract: Human–mouse comparative genomics is an informative tool to assess sequence functionality as inferred from its conservation level. We used this approach to examine dependency among different positions of the 5′ splice site. We compiled a data set of 50,493 homologous human–mouse internal exons and analyzed the frequency of changes among different positions of homologous human–mouse 5′ splice-site pairs. We found mutual relationships between positions +4 and +5, +5 and +6, −2 and +5, and −1 and +5. We also demonstrated the association between the exonic and the intronic positions of the 5′ splice site, in which a stronger interaction of U1 snRNA and the intronic portion of the 5′ splice site compensates for weak interaction of U1 snRNA and the exonic portion of the 5′ splice site, and vice versa. By using an ex vivo system that mimics the effect of mutation in the 5′ splice site leading to familial dysautonomia, we demonstrated that U1 snRNA base-pairing with positions +6 and −1 is the only functional requirement for mRNA splicing of this 5′ splice site. Our findings indicate the importance of U1 snRNA base-pairing to the exonic portion of the 5′ splice site.
TL;DR: The observed evolutionary conservation of GORS in many viruses imposes a considerable constraint on genome plasticity and the consequent narrowing of sequence space in which neutral drift can occur, potentially reconcile the rapid evolution of RNA viruses over short periods with the documented examples of extreme conservatism evident from their intimate coevolution with their hosts.
Abstract: Discrete RNA secondary and higher-order structures, typically local in extent, play a fundamental role in RNA virus replication. Using new bioinformatics analysis methods, we have identified genome-scale ordered RNA structure (GORS) in many genera and families of positive-strand animal and plant RNA viruses. There was remarkably variability between genera that possess this characteristic; for example, hepaciviruses in the family Flaviviridae show evidence for extensive internal base-pairing throughout their coding sequences that was absent in both the related pestivirus and flavivirus genera. Similar genus-associated variability was observed in the Picornaviridae, the Caliciviridae, and many plant virus families. The similarity in replication strategies between genera in each of these families rules out a role for GORS in a fundamentally conserved aspect of this aspect of the virus life cycle. However, in the Picornaviridae, Flaviviridae, and Caliciviridae, the existence of GORS correlated strongly with the ability of each genus to persist in their natural hosts. This raises the intriguing possibility of a role for GORS in the modulation of innate intracellular defense mechanisms (and secondarily, the acquired immune system) triggered by double-stranded RNA, analogous in function to the expression of structured RNA transcripts by large DNA viruses. Irrespective of function, the observed evolutionary conservation of GORS in many viruses imposes a considerable constraint on genome plasticity and the consequent narrowing of sequence space in which neutral drift can occur. These findings potentially reconcile the rapid evolution of RNA viruses over short periods with the documented examples of extreme conservatism evident from their intimate coevolution with their hosts.
TL;DR: Evidence is presented that a component of the cleavage-polyadenylation specificity factor CPSF-73 is the long-sought endonuclease, and it is shown that 3' cleavage is metal-dependent, likely reflecting a requirement for tightly protein-bound Zn(2+).
Abstract: Generation of the polyadenylated 3 end of an mRNA requires an endonucleolytic cleavage followed by synthesis of the poly(A) tail. Despite the seeming simplicity of the reaction, more than a dozen polypeptides are required, and nearly all appear to be necessary for the cleavage reaction. Because of this complexity, the identity of the endonuclease has remained a mystery. Here we present evidence that a component of the cleavage-polyadenylation specificity factor CPSF-73 is the long-sought endonuclease. We first show, using site-specific labeling and UV-cross-linking, that a protein with properties of CPSF-73 is one of only two polypeptides in HeLa nuclear extract to contact the cleavage site in an AAUAAA-dependent manner. The recent identification of CPSF-73 as a possible member of the metallo--lactamase family of Zn 2+ -dependent hydrolytic enzymes suggests that this contact may identify CPSF-73 as the nuclease. Supporting the significance of the putative hydrolytic lactamase domain in CPSF-73, we show that mutation of key residues predicted to be required for activity in the yeast CPSF-73 homolog result in lethality. Furthermore, in contrast to long held belief, but consistent with properties of metallo--lactamases, we show that 3 cleavage is metal-dependent, likely reflecting a requirement for tightly protein-bound Zn 2+ . Taken together, the available
TL;DR: It is found that relatively long, polyadenylated transcripts encoded by the Caenorhabditis elegans let-7 gene undergo trans-splicing to the spliced leader 1 (SL1) RNA.
Abstract: Members of the microRNA (miRNA) class of 22-nucleotide RNAs regulate the expression of target genes that contain sequences of antisense complementarity. Maturation of miRNAs involves cleavage of longer primary transcripts, but little is yet understood about how miRNA genes are transcribed and enter the processing pathway. We find that relatively long, polyadenylated transcripts encoded by the Caenorhabditis elegans let-7 gene undergo trans-splicing to the spliced leader 1 (SL1) RNA. Deletions, including removal of the trans-splice site, upstream of mature let-7 sequence result in stable accumulation of primary transcripts and compromised production of mature let-7 RNA in vivo. Our data show that multiple steps of let-7 miRNA biogenesis can be uncoupled, allowing for complex regulation in the production of a functional miRNA. Finally, the observation that let-7 primary transcripts undergo splicing highlights the importance of identifying the sequence of endogenous pri-miRNA substrates recognized by the cellular processing machinery.
TL;DR: The analysis revealed that the mRNA destabilization associated with NMD could be separated into two distinct forms that correlated with the extent the premature stop codon was suppressed, suggesting that Upf1p may associate with the termination complex prior to polypeptide chain release.
Abstract: The Nonsense-Mediated mRNA Decay (NMD) pathway mediates the rapid degradation of mRNAs that contain premature stop mutations in eukaryotic organisms. It was recently shown that mutations in three yeast genes that encode proteins involved in the NMD process, UPF1, UPF2, and UPF3, also reduce the efficiency of translation termination. In the current study, we compared the efficiency of translation termination in a upf1Delta strain and a [PSI(+)] strain using a collection of translation termination reporter constructs. The [PSI(+)] state is caused by a prion form of the polypeptide chain release factor eRF3 that limits its availability to participate in translation termination. In contrast, the mechanism by which Upf1p influences translation termination is poorly understood. The efficiency of translation termination is primarily determined by a tetranucleotide termination signal consisting of the stop codon and the first nucleotide immediately 3' of the stop codon. We found that the upf1Delta mutation, like the [PSI(+)] state, decreases the efficiency of translation termination over a broad range of tetranucleotide termination signals in a unique, context-dependent manner. These results suggest that Upf1p may associate with the termination complex prior to polypeptide chain release. We also found that the increase in readthrough observed in a [PSI(+)]/upf1Delta strain was larger than the readthrough observed in strains carrying either defect alone, indicating that the upf1Delta mutation and the [PSI(+)] state influence the termination process in distinct ways. Finally, our analysis revealed that the mRNA destabilization associated with NMD could be separated into two distinct forms that correlated with the extent the premature stop codon was suppressed. The minor component of NMD was a 25% decrease in mRNA levels observed when readthrough was >/=0.5%, while the major component was represented by a larger decrease in mRNA abundance that was observed only when readthrough was
TL;DR: It is found that with the loops in place, folding into the active conformation occurs in a single step, in the microM range of magnesium ion concentration.
Abstract: It has been shown that the activity of the hammerhead ribozyme at μM magnesium ion concentrations is markedly increased by the inclusion of loops in helices I and II. We have studied the effect of such loops on the magnesium ion-induced folding of the ribozyme, using fluorescence resonance energy transfer. We find that with the loops in place, folding into the active conformation occurs in a single step, in the μM range of magnesium ion concentration. Disruption of the loop–loop interaction leads to a reversion to two-step folding, with the second stage requiring mM concentrations of magnesium ion. Sodium ions also promote the folding of the natural form of the ribozyme at high concentrations, but the folding occurs as a two-stage process. The loops clearly act as important auxiliary elements in the function of the ribozyme, permitting folding to occur efficiently under physiological conditions.
TL;DR: The combined use of a new RNAi-based method together with RT-PCR to effectively identify aberrant RNAs is proposed and stressed the importance of using alternative test procedures in conjunction with a combination of sensitive RNA analyses for discerning IRES-containing sequences in eukaryotic mRNAs.
Abstract: The dicistronic assay for internal ribosome entry site (IRES) activity is the most widely used method for testing putative sequences that may drive cap-independent translation initiation. This assay typically involves the transfection of cells with dicistronic DNA test constructs. Many of the reports describing eukaryotic IRES elements have been criticized for the use of inadequate methods for the detection of aberrant RNAs that may form in transfected cells using this assay. Here we propose the combined use of a new RNAi-based method together with RT-PCR to effectively identify aberrant RNAs. We illustrate the use of these methods for analysis of RNAs generated in cells transfected with dicistronic test DNAs containing either the hepatitis C virus (HCV) IRES or the X-linked inhibitor of apoptosis (XIAP) cellular IRES. Both analyses indicated aberrantly spliced transcripts occurred in cells transfected with the XIAP dicistronic DNA construct. This contributed to the unusually high levels of apparent IRES activity exhibited by the XIAP 5′ UTR in vivo. Cells transfected directly with dicistronic RNA exhibited much lower levels of XIAP IRES activity, resembling the lower levels observed after translation of dicistronic RNA in rabbit reticulocyte lysates. No aberrantly spliced transcripts could be detected following direct RNA transfection of cells. Interestingly, transfection of dicistronic DNA or RNA containing the HCV IRES did not form aberrantly spliced transcripts. These observations stress the importance of using alternative test procedures (e.g., direct RNA transfection) in conjunction with a combination of sensitive RNA analyses for discerning IRES-containing sequences in eukaryotic mRNAs.
TL;DR: The results show that the tightly kinked conformation of the K-turn requires stabilization by other factors, possibly by protein binding, for example, and is therefore unlikely to be of itself a primary organizing feature in RNA.
Abstract: The kink-turn (K-turn) is a new motif in RNA structure that was identified by examination of the crystal structures of the ribosome. We examined the structural and dynamic properties of this element in free solution. The K-turn RNA exists in a dynamic equilibrium between a tightly kinked conformation and a more open structure similar to a simple bulge bend. The highly kinked form is stabilized by the noncooperative binding of metal ions, but a significant population of the less-kinked form is present even in the presence of relatively high concentrations of divalent metal ions. The conformation of the tightly kinked population is in excellent agreement with that of the K-turn structures observed in the ribosome by crystallography. The end-to-end FRET efficiency of this species agrees closely with that of the ribosomal K-turn, and the direction of the bend measured by comparative gel electrophoresis also corresponds very well. These results show that the tightly kinked conformation of the K-turn requires stabilization by other factors, possibly by protein binding, for example. The K-turn is therefore unlikely to be of itself a primary organizing feature in RNA.
TL;DR: The results suggest that the evolutionary conserved weak 5' ss may serve as a mechanism to regulate exon 7 splicing under different physiological contexts, and are the first report in which a functional selection method has been applied to analyze the entire exon.
Abstract: Humans have two near identical copies of the survival of motor neuron (SMN) gene, SMN1 and SMN2. In spinal muscular atrophy (SMA), SMN2 is not able to compensate for the loss of SMN1 due to an inhibitory mutation at position 6 (C6U mutation in transcript) of exon 7. We have recently shown that C6U creates an extended inhibitory context (Exinct) that causes skipping of exon 7 in SMN2. Previous studies have shown that an exonic splicing enhancer associated with Tra2 (Tra2-ESE) is required for exon 7 inclusion in both SMN1 and SMN2. Here we describe the method of in vivo selection that determined the position-specific role of wild-type nucleotides within the entire exon 7. Our results confirmed the existence of Exinct and revealed the presence of an additional inhibitory tract (3'-Cluster) near the 3'-end of exon 7. We also demonstrate that a single nucleotide substitution at the last position of exon 7 improves the 5' splice site (ss) such that the presence of inhibitory elements (Exinct as well as the 3'-Cluster) and the absence of Tra2-ESE no longer determined exon 7 usage. Our results suggest that the evolutionary conserved weak 5' ss may serve as a mechanism to regulate exon 7 splicing under different physiological contexts. This is the first report in which a functional selection method has been applied to analyze the entire exon. This method offers unparallel advantage for determining the relative strength of splice sites, as well as for identifying the novel exonic cis-elements.
TL;DR: In this paper, the authors have identified the protein components of U11/U12 snRNPs by using mass spectrometry and showed that within the U12-type prespliceosome, U11 and U12 components form a molecular bridge connecting both ends of the intron.
Abstract: U11 and U12 snRNPs bind U12-type pre-mRNAs as a preformed di-snRNP complex, simultaneously recognizing the 5 splice site and branchpoint sequence. Thus, within the U12-type prespliceosome, U11/U12 components form a molecular bridge connecting both ends of the intron. We have affinity purified human 18S U11/U12 and 12S U11 snRNPs, and identified their protein components by using mass spectrometry. U11/U12 snRNPs lack all known U1 snRNP proteins but contain seven novel proteins (i.e., 65K, 59K, 48K, 35K, 31K, 25K, 20K) not found in the major spliceosome, four of which (59K, 48K, 35K, and 25K) are U11-associated. Thus, protein–protein and protein–RNA interactions contributing to 5 splice site recognition and/or intron bridging appear to differ significantly in the minor versus major prespliceosome. The majority of U11/U12 proteins are highly conserved in organisms known to contain U12-type introns. However, homologs of those associated with U11 were not detected in Drosophila melanogaster, consistent with the presence of a divergent U11 snRNP in flies. RNAi experiments revealed that several U11/U12 proteins are essential for cell viability, suggesting they play key roles in U12-type splicing. The presence of unique U11/U12 snRNP proteins in the U12-type spliceosome provides insight into potential evolutionary relationships between the major and minor spliceosome.
TL;DR: In vitro interaction studies performed in the presence and absence of RanQ69LGTP indicate that all three transportins most likely act as import factors for HuR and hnRNP A1, and possible nucleocytoplasmic shuttling mechanisms for hnGTP A1 and HuR are discussed.
Abstract: Several mRNA-binding proteins, including hnRNP A1 and HuR, contain bidirectional transport signals that mediate both their nuclear import and export. Previously, Transportin 1 (Trn1) was identified as a mediator of hnRNP A1 import, whereas the closely related protein Transportin 2 (Trn2) was shown to interact with HuR. Here we have investigated the subfamily of transportins that consists of Trn1 (or Kap β2A) and two alternatively spliced Trn2 isoforms (Trn2a and Trn2b), also called Trn2 and Kap β2B. The sequence differences among these proteins could alter either their cargo specificity or their response to RanGTP and thus their function as import or export receptors. Using in vitro binding assays, we show that hnRNP A1 preferentially binds Trn1 and Trn2b versus Trn2a. HuR interacts with all three transportins, as well as weakly with Imp β. The hnRNP A1 and HuR shuttling domains, called M9 and HNS, respectively, are sufficient for these interactions. Despite small differences in the binding of HuR and hnRNP A1 to the three transportins, in vitro interaction studies performed in the presence and absence of RanQ69LGTP indicate that all three transportins most likely act as import factors for HuR and hnRNP A1. In digitonin-permeabilized HeLa cells, both M9 and HNS peptides compete for the import of recombinant hnRNP A1 and HuR, indicating that HuR and hnRNP A1 import pathways are at least partially overlapping. Possible nucleocytoplasmic shuttling mechanisms for hnRNP A1 and HuR are discussed.