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Alternative splicing of HLA-DQB1 exon 4 is allele-dependent and results in variable expression of soluble DQβ. We have recently shown that differential inclusion of this exon in mature transcripts is largely due to intron 3 variants in the branch point sequence (BPS) and polypyrimidine tract. To identify additional regulatory cis-elements that contribute to haplotype-specific splicing of DQB1, we systematically examined the effect of guanosine (G) repeats on intron 3 removal. We found that the GGG or GGGG repeats generally improved splicing of DQB1 intron 3, except for those that were adjacent to the 5′ splice site where they had the opposite effect. The most prominent splicing enhancement was conferred by GGGG motifs arranged in tandem upstream of the BPS. Replacement of a G-rich segment just 5′ of the BPS with a series of random sequences markedly repressed splicing, whereas substitutions of a segment further upstream that lacked the G-rich elements and had the same size did not result in comparable splicing inhibition. Systematic mutagenesis of both suprabranch guanosine quadruplets (G4) revealed a key role of central G residues in splicing enhancement, whereas cytosines in these positions had the most prominent repressive effects. Together, these results show a significant role of tandem G4NG4 structures in splicing of both complete and truncated DQB1 intron 3, support position dependency of G repeats in splicing promotion and inhibition, and identify positively and negatively acting sequences that contribute to the haplotype-specific DQB1 expression.

Purpose: Most of the kits or reagents available for testing for mutations in the cystic fibrosis transmembrane conductance regulator gene include testing for the 5/7/9T polypyrimidine tract, but these methods only screen for three variants in this region: 5T, 7T, and 9T. Although such commercial products may not have been designed to screen for rare alleles of the polypyrimidine tract, we demonstrate that at least one of them (Tag-It™ Cystic Fibrosis Kit, Tm Bioscience, Toronto, Ontario, Canada) has enough sensitivity to differentiate samples with rare alleles by describing how this product allowed us to detect a previously uncharacterized 11T allele. Methods: A total of 139 banked and anonymized clinical samples from carrier adults and children with cystic fibrosis (The Hospital for Sick Children, Toronto, Canada) were tested and analyzed using the Tag-It™ Cystic Fibrosis Kit. Results: Two samples displayed allelic ratios for the polypyrimidine tract that were significantly different from the other samples and did not correspond to values expected to be seen for samples with 5T, 7T, or 9T alleles. Further tests with sequencing and an extended Tag-It™ assay confirmed the presence of an 11T allele. Conclusion: Although commercial products used in cystic fibrosis testing may not have been designed to screen for rare alleles of the polypyrimidine tract, we demonstrated that at least the Tag-It™ assay may have enough sensitivity to differentiate samples with such rare alleles, which can then be further analyzed for clarification.

Human alternative isoform, cryptic, skipped, and constitutive splice sites from the ALTEXTRON database were analysed regarding splice site strength, composition, GC content, position and binding site strength of polypyrimidine tract and branch site. Several features were identified which distinguish alternative isoform and cryptic splice sites, but not skipped splice sites from constitutive ones. These include splice site strength, introns GC content, U2AF35 binding site score, and oligonucleotide frequencies. For the predictive classification of splice sites, pattern recognition models for different splicing factor binding sites and oligonucleotide frequency models (OFMs) were combined using backpropagation networks. 67.45% of acceptor sites and 71.23% of donor sites are correctly classified by networks trained for classification of constitutive and alternative isoform/cryptic splice sites. A web-application for the prediction of alternative splice sites is available at http://es.embnet.org/~mwang/assp.html .

The carboxy-terminal domain (CTD) of the large subunit of RNA polymerase II (pol II) is essential for several co-transcriptional pre-messenger RNA processing events, including capping, 3′-end processing and splicing. We investigated the role of the CTD of RNA pol II in the coordination of A to I editing and splicing of the ADAR2 (ADAR: adenosine deaminases that act on RNA) pre-mRNA. The auto-editing of Adar2 intron 4 by the ADAR2 adenosine deaminase is tightly coupled to splicing, as the modification of the dinucleotide AA to AI creates a new 3′ splice site. Unlike other introns, the CTD is not required for efficient splicing of intron 4 at either the normal 3′ splice site or the alternative site created by editing. However, the CTD is required for efficient co-transcriptional auto-editing of ADAR2 intron 4. Our results implicate the CTD in site-selective RNA editing by ADAR2 and in coordination of editing with alternative splicing.

Polypyrimidine tract-binding protein (PTB) is an hnRNP with four RRM type domains. It plays roles as a repressive alternative splicing regulator of multilple target genes, as well as being involved in pre-mRNA 3' end processing, mRNA localization, stability, and internal ribosome entry site-mediated translation. Here we have used a tethered function assay, in which a fusion protein of PTB and the bacteriophage MS2 coat protein is recruited to a splicing regulatory site by binding to an artificially inserted MS2 binding site. Deletion mutations of PTB in this system allowed us to identify RRM2 and the following inter-RRM linker region as the minimal region of PTB that can act as splicing repressor domain when recruited to RNA. Splicing repression by the minimal repressor domain remained cell type-specific and dependent upon other defined regulatory elements in the alpha-tropomyosin test minigene. Our results highlight the fact that splicing repression by PTB can be uncoupled from the mode by which it binds to RNA.

Background The 3′ splice site (SS) at the end of pre-mRNA introns has a consensus sequence (Y)nNYAG for constitutive splicing of mammalian genes. Deviation from this consensus could change or interrupt the usage of the splice site leading to alternative or aberrant splicing, which could affect normal cell function or even the development of diseases. We have shown that the position “N” can be replaced by a CA-rich RNA element called CaRRE1 to regulate the alternative splicing of a group of genes. Results Taking it a step further, we searched the human genome for purine-rich elements between the -3 and -10 positions of the 3′ splice sites of annotated introns. This identified several thousand such 3′SS; more than a thousand of them contain at least one copy of G tract. These sites deviate significantly from the consensus of constitutive splice sites and are highly associated with alterative splicing events, particularly alternative 3′ splice and intron retention. We show by mutagenesis analysis and RNA interference that the G tracts are splicing silencers and a group of the associated exons are controlled by the G tract binding proteins hnRNP H/F. Species comparison of a group of the 3′SS among vertebrates suggests that most (~87%) of the G tracts emerged in ancestors of mammals during evolution. Moreover, the host genes are most significantly associated with cancer. Conclusion We call these elements together with CaRRE1 regulatory RNA elements between the Py and 3′AG (REPA). The emergence of REPA in this highly constrained region indicates that this location has been remarkably permissive for the emergence of de novo regulatory RNA elements, even purine-rich motifs, in a large group of mammalian genes during evolution. This evolutionary change controls alternative splicing, likely to diversify proteomes for particular cellular functions. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-1143) contains supplementary material, which is available to authorized users.

The spliceosome, the gigantic molecular machine that performs pre-mRNA splicing in eukaryotes, contains over 200 different proteins and five RNA molecules. The central role played by the spliceosomal RNAs in splicing has led to the hypothesis that, like the ribosome, the spliceosome is an RNA-centric enzyme and a relic from the RNA world. Recent structural studies have provided the first glimpses of the structural features of spliceosomal RNAs, and mutational analyses in vivo and in vitro have uncovered new functional roles for a catalytically essential domain. An emerging model for the active site of group II introns, a closely related class of natural ribozymes, is likely to provide a wealth of insights on structure and function of the active site of the spliceosome.

The promoter of the hypoxia inducible factor 1 alpha (HIF-1alpha) gene has a polypurine/polypyrimidine tract (-65 to -85) overlapping or adjacent to several putative transcription factor binding sites, and we found that mutagenesis of this region diminished basal HIF-1alpha expression. Oligonucleotides representing this region of the HIF-1alpha promoter were analyzed by electrophoretic mobility shift, chemical probing, circular dichroism, and DNA polymerase arrest assays. The guanine-rich strand was found to form a parallel, unimolecular quadruplex in the presence of potassium that was further stabilized by two known quadruplex binding compounds, the cationic porphyrin TmPyP4 and the natural product telomestatin, while TmPyP2, a positional isomer of TmPyP4, did not stabilize quadruplex formation. These data suggest that a quadruplex structure may form in a region of the HIF-1alpha promoter that regulates basal HIF-1alpha expression.

The proximal promoter region of the human vascular endothelial growth factor (VEGF) gene contains a polypurine/polypyrimidine tract that serves as a multiple binding site for Sp1 and Egr-1 transcription factors. This tract contains a guanine-rich sequence consisting of four runs of three or more contiguous guanines separated by one or more bases, corresponding to a general motif for the formation of an intramolecular G-quadruplex. In this study, we observed the progressive unwinding of the oligomer duplex DNA containing this region into single-stranded forms in the presence of KCl and the G-quadruplex-interactive agents TMPyP4 and telomestatin, suggesting the dynamic nature of this tract under conditions which favor the formation of the G-quadruplex structures. Subsequent footprinting studies with DNase I and S1 nucleases using a supercoiled plasmid DNA containing the human VEGF promoter region also revealed a long protected region, including the guanine-rich sequences, in the presence of KCl and telomestatin. Significantly, a striking hypersensitivity to both nucleases was observed at the 3′-side residue of the predicted G-quadruplex-forming region in the presence of KCl and telomestatin, indicating altered conformation of the human VEGF proximal promoter region surrounding the guanine-rich sequence. In contrast, when specific point mutations were introduced into specific guanine residues within the G-quadruplex-forming region (Sp1 binding sites) to abolish G-quadruplex-forming ability, the reactivity of both nucleases toward the mutated human VEGF proximal promoter region was almost identical, even in the presence of telomestatin with KCl. This comparison of wild-type and mutant sequences strongly suggests that the formation of highly organized secondary structures such as G-quadruplexes within the G-rich region of the human VEGF promoter region is responsible for observed changes in the reactivity of both nucleases within the polypurine/polypyrimidine tract of the human VEGF gene. The formation of the G-quadruplex structures from this G-rich sequence in the human VEGF promoter is further confirmed by the CD experiments. Collectively, our results provide strong evidence that specific G-quadruplex structures can naturally be formed by the G-rich sequence within the polypurine/polypyrimidine tract of the human VEGF promoter region, raising the possibility that the transcriptional control of the VEGF gene can be modulated by G-quadruplex-interactive agents.

We compiled sequences of previously published aberrant 3 0 splice sites (3 0 ss) that were generated by mutations in human disease genes. Cryptic 3 0 ss, defined here as those resulting from a mutation of the 3 0 YAG consensus, were more frequent in exons than in introns. They clustered in � 20 nt region adjacent to authentic 3 0 ss, suggesting that their underrepresentation in introns is due to a depletion of AG dinucleotides in the polypyrimidine tract (PPT). In contrast, most aberrant 3 0 ss that were induced by mutations outside the 3 0 YAG consensus (designated ‘de novo’) were in introns. The activation of intronic de novo 3 0 ss was largely due to AG-creating mutations in the PPT. In contrast, exonic de novo 3 0 ss were more often induced by mutations improving the PPT, branchpoint sequence (BPS) or distant auxiliary signals, rather than by direct AG creation. The Shapiro– Senapathy matrix scores had a good prognostic value for cryptic, but not de novo 3 0 ss. Finally, AG-creating mutations in the PPT that produced aberrant 3 0 ss upstream of the predicted BPS in vivo shared a similar ‘BPS-new AG’ distance. Reduction of this distance and/or the strength of the new AG PPT in splicing reporter pre-mRNAs improved utilization of authentic 3 0 ss, suggesting that AG-creating mutations that are located closer to the BPS and are preceded by weaker PPT may result in less severe splicing defects.

Polypyrimidine tract binding protein (PTB) represses the splicing of many alternatively spliced exons. This repression can sometimes involve the direct occlusion of splice sites by PTB. We show here that PTB prevents splicing of the c-src N1 exon to downstream exon 4 by a different mechanism. PTB does not interfere with U1 snRNP binding to the N1 5′ splice site, but instead prevents formation of the pre-spliceosomal Early (E) complex across the intervening intron. If only the repressed 5′ splice site of the N1 exon is present, the splicing factor U2AF does not assemble on the downstream 3′ splice site of exon 4. When the unregulated 5′ splice site of the upstream exon 3 is included in the RNA, U2AF binding is restored and splicing between exons 3 and 4 proceeds, in spite of the presence of the PTB bound across the N1 exon. Rather than directly blocking the N1 splice sites, PTB is blocking the 5′ splice site dependent assembly of U2AF into the E complex, presumably through an interaction with the U1 snRNP. This mechanism of repression is likely to also occur in many other alternative exons.

We have analyzed the interaction between the U2AF subunits U2AF35 and U2AF65 in vivo using fluorescence resonance energy transfer (FRET) microscopy. U2 snRNP Auxiliary Factor (U2AF) is an essential pre-mRNA splicing factor complex, comprising 35-kDa (U2AF35) and 65-kDa (U2AF65) subunits. U2AF65 interacts directly with the polypyrimidine tract and promotes binding of U2 snRNP to the pre-mRNA branchpoint, while U2AF35 associates with the conserved AG dinucleotide at the 3′ end of the intron and has multiple functions in the splicing process. Using two different approaches for measuring FRET, we have identified and spatially localized sites of direct interaction between U2AF35 and U2AF65 in vivo in live cell nuclei. While U2AF is thought to function as a heterodimeric complex, the FRET data have also revealed a novel U2AF35 self-interaction in vivo, which is confirmed in vitro using biochemical assays. These results suggest that the stoichiometry of the U2AF complex may, at least in part, differ in vivo from the expected heterodimeric complex. The data show that FRET studies offer a valuable approach for probing interactions between pre-mRNA splicing factors in vivo.

Foot-and-mouth disease (FMD), the most economically important disease of cloven-hoofed animals, is endemic in India. Sequence analysis revealed that phylogenetic grouping of type Asia1 field isolates on the basis of the large fragment of the 5'untranslated region (5'LF-UTR) was quite similar to that based on the sequences of the capsid-coding (VP1) region of the same viruses. The existence of two distinct lineages of type Asia1 suggested by the study on the VP1 region was further supported by the detection of a difference in length and predicted secondary structure of the 5'LF-UTR between the two lineages. Sequence variability between the isolates of the two lineages was also observed within the different domains of the internal ribosome entry site (IRES) around conserved motifs like the GNRA,- RAAA,- and the polypyrimidine tract. Certain group and lineage-specific signature nucleotides pertaining to FMDV type Asia1 in the 5'LF-UTR have been identified. The present study shows that the 5'LF-UTR of FMDV serotype Asia1 field isolates are variable in relation to the length and probable secondary structure of the IRES.

Pre-messenger RNA (pre-mRNA) splicing is a central step in gene expression. Lying between transcription and protein synthesis, pre-mRNA splicing removes sequences (introns) that would otherwise disrupt the coding potential of intron-containing transcripts. This process takes place in the nucleus, catalyzed by a large RNA–protein complex called the spliceosome. Prp8p, one of the largest and most highly conserved of nuclear proteins, occupies a central position in the catalytic core of the spliceosome, and has been implicated in several crucial molecular rearrangements that occur there. Recently, Prp8p has also come under the spotlight for its role in the inherited human disease, Retinitis Pigmentosa.Prp8 is unique, having no obvious homology to other proteins; however, using bioinformatical analysis we reveal the presence of a conserved RNA recognition motif (RRM), an MPN/JAB domain and a putative nuclear localization signal (NLS). Here, we review biochemical and genetical data, mostly related to the human and yeast proteins, that describe Prp8’s central role within the spliceosome and its molecular interactions during spliceosome formation, as splicing proceeds, and in post-splicing complexes.

Hepatitis C virus (HCV) genome contains a 3'noncoding region (3'NCR) consisting of a variable region, a polypyrimidine tract (polyU/UC) and the X region. To examine the roles of 3'NCR and polyU/UC tract in the internal ribosome entry site (IRES)-mediated translation process, a variety of 3'NCRs containing different lengths of polyU/UC tract were obtained from HCV infected patients and cloned respectively to the downstream of the firefly luciferase coding gene linked to HCV 5'NCR and 30 nucleotides of core gene (containing IRES element). The results of in vitro translation in rabbit reticulocyte lysate (RRL) and cell transfection assay in mammalian cells showed that the IRES-mediated translation efficiency could be enhanced by the full-length of 3'NCR of HCV RNA. However, contradictory results were observed when the role of polyU/UC tract in the IRES-mediated translation was studied. While the IRES-mediated translation efficiency was inhibited by the presence of polyU/UC tract in in vitro translation experiments, transfection of these expression cassettes into hepatic cell line showed that polyU/UC tract enhanced IRES-mediated translation efficiency in vivo. Cellular-fraction complement experiments showed that cellular factors were required for the enhancement by the polyU/UC tract. Further antibody blocking assay and UV cross-linking assay suggested the correlation of IRES-mediated translation with host factors, including the La protein. The data above also indicated that the modulations of the IRES-mediated translation by the HCV 3'NCR and the polyU/UC tract were in a length-independent manner.

Transcription of the HIV-1 genome yields a single primary transcript, which is alternatively spliced to >30 mRNAs. Productive infection depends on inefficient and regulated splicing and appears to proceed in a tight 5' to 3' order. To analyse whether sequential splicing is mediated by the quality of splice sites or by the position of an intron, we inserted the efficient beta-globin intron (BGI) into the 3' region or 5'UTR of a subgenomic expression vector or an infectious proviral plasmid. RNA analysis revealed splicing of the 3' BGI only if all upstream introns were removed, while splicing of the same intron in the 5'UTR was efficient and independent of further splicing. Furthermore, mutation of the upstream splice signal in the subgenomic vector did not eliminate the inhibition of 3' splicing, although the BGI sequence was the only intron in this case. These results suggest that downstream splicing of HIV-1 RNAs is completely dependent on prior splicing of all upstream intron(s). This hypothesis was supported by the mutation of the major 5' splice site in the HIV-1 genome, which completely abolished all splicing. It appears likely that the tight order of splicing is important for HIV-1 replication, which requires the stable production of intron containing RNAs, while splicing of 3' introns on incompletely spliced RNAs would be likely to render them subject to nonsense-mediated decay.

The perinucleolar compartment (PNC) is a sub-nuclear structure that preferentially localizes to the nucleolar periphery. The PNC is found predominantly in transformed cells both in vitro and in vivo. PNC prevalence (the percentage of cells containing at least one PNC) positively correlates with the progression of breast cancer and patient survival. PNCs are highly enriched with newly synthesized RNA polymerase III transcripts and RNA-binding proteins. The structural integrity of the PNC is dependent upon the transcription of these RNAs and a critical level of the polypyrimidine tract binding (PTB) protein, as assayed by the localization of other PNC-associated proteins. These observations suggest a model in which the PNC is a dynamic, functional organelle that forms under specific physiological conditions favoring cellular transformation and might be involved in the metabolism of RNA polymerase III transcripts.

Peutz-Jeghers syndrome (PJS) is an autosomal dominant disorder associated with gastrointestinal polyposis and an increased cancer risk. PJS is caused by germline mutations in the tumor suppressor gene LKB1. One such mutation, IVS2+1A>G, alters the second intron 5' splice site, which has sequence features of a U12-type AT-AC intron. We report that in patients, LKB1 RNA splicing occurs from the mutated 5' splice site to several cryptic, noncanonical 3' splice sites immediately adjacent to the normal 3' splice site. In vitro splicing analysis demonstrates that this aberrant splicing is mediated by the U12-dependent spliceosome. The results indicate that the minor spliceosome can use a variety of 3' splice site sequences to pair to a given 5' splice site, albeit with tight constraints for maintaining the 3' splice site position. The unusual splicing defect associated with this PJS-causing mutation uncovers differences in splice-site recognition between the major and minor pre-mRNA splicing pathways.

Pre-mRNA splicing in eukaryotes requires joining together the nucleotides of the various mRNA-coding regions (exons) after recognizing them from the normally vastly superior number of non-mRNA-coding sequences (introns). For three excellent reviews on general splicing and its regulation, refer to references 14, 62, and 70. In eukaryotes, the vast majority of splicing processes are catalyzed by the spliceosome, a very complex RNA-protein aggregate which has been estimated to contain several hundred different proteins in addition to five spliceosomal snRNAs (1, 54, 62, 63, 81, 109). These factors are responsible for the accurate positioning of the spliceosome on the 5′ and 3′ splice site sequences. The reason why so many factors are needed reflects the observation that exon recognition can be affected by many pre-mRNA features such as exon length (5, 97), the presence of enhancer and silencer elements (8, 62), the strength of splicing signals (45), the promoter architecture (29, 55), and the rate of RNA processivity (86). In addition, the general cellular environment also exerts an effect, as recent observations suggest the existence of extensive coupling between splicing and many other gene expression steps (69) and even its modification by external stimuli (96).

The approximately 1.2-kb 5'-noncoding region (5'-NCR) of mRNA species encoding mouse Kv1.4, a member of the Shaker-related subfamily of voltage-gated potassium channels, was shown to mediate internal ribosome entry in cells derived from brain, heart, and skeletal muscle, tissues known to express Kv1.4 mRNA species. We also show that the upstream approximately 1.0 kb and the downstream approximately 0.2 kb of the Kv1.4 5'-NCR independently mediated internal ribosome entry; however, separately, these sequences were less efficient in mediating internal ribosome entry than when together in the complete (and contiguous) 5'-NCR. Using enzymatic structure probing, the 3'-most approximately 0.2 kb was predicted to form three distinct stem-loop structures (stem-loops X, Y, and Z) and two defined single-stranded regions (loops Psi and Omega) in the presence and absence of the upstream approximately 1.0 kb. Although the systematic deletion of sequences within the 3'-most approximately 0.2 kb resulted in distinct changes in expression, enzymatic structure probing indicated that local RNA folding was not completely altered. Structure probing analysis strongly suggested an interaction between stem-loop X and a downstream polypyrimidine tract; however, opposing changes in activity were observed when sequences within these two regions were independently deleted. Moreover, deletions correlating with positive as well as negative changes in expression altered RNase cleavage within stem-loop X, indicating that this structure may be an integral element. Therefore, these findings indicate that Kv1.4 expression is mediated through a complex interplay between many distinct RNA regions.

We have previously identified cis -acting RNA sequences in the human papillomavirus type 16 (HPV-16) L1 coding region which inhibit expression of L1 from eukaryotic expression plasmids. Here we have determined the function of one of these RNA elements, and we provide evidence that this RNA element is a splicing silencer which suppresses the use of the 3′ splice site located immediately upstream of the L1 AUG. We also show that this splice site is inefficiently utilized as a result of a suboptimal polypyrimidine tract. Introduction of point mutations in the L1 coding region that altered the RNA sequence without affecting the L1 protein sequence resulted in the inactivation of the splicing silencer and induced splicing to the L1 3′ splice site. These mutations also prevented the interaction of the RNA silencer with a 35-kDa cellular protein identified here as hnRNP A1. The splicing silencer in L1 inhibits splicing in vitro, and splicing can be restored by the addition of RNAs containing an hnRNP A1 binding site to the reaction, demonstrating that hnRNP A1 inhibits splicing of the late HPV-16 mRNAs through the splicing silencer sequence. While we show that one role of the splicing silencer is to determine the ratio between partially spliced L2/L1 mRNAs and spliced L1 mRNAs, we also demonstrate that it inhibits splicing from the major 5′ splice site in the early region to the L1 3′ splice site, thereby playing an essential role in preventing late gene expression at an early stage of the viral life cycle. We speculate that the activity of the splicing silencer and possibly the concentration of hnRNP A1 in the HPV-16-infected cell determines the ability of the virus to establish a persistent infection which remains undetected by the host immune surveillance.

The protein factor U2AF is an essential component required for pre-mRNA splicing. Mutations identified in the S. pombe large U2AF subunit were used to engineer transgenic Drosophila carrying temperature-sensitive U2AF large subunit alleles. Mutant recombinant U2AF heterodimers showed reduced polypyrimidine tract RNA binding at elevated temperatures. Genome-wide RNA profiling comparing wild-type and mutant strains identified more than 400 genes differentially expressed in the dU2AF50 mutant flies grown at the restrictive temperature. Surprisingly, almost 40% of the downregulated genes lack introns. Microarray analyses revealed that nuclear export of a large number of intronless mRNAs is impaired in Drosophila-cultured cells RNAi knocked down for dU2AF50. Immunopurification of nuclear RNP complexes showed that dU2AF50 associates with intronless mRNAs. These results reveal an unexpected role for the splicing factor dU2AF50 in the nuclear export of intronless mRNAs.

Splicing of mouse immunoglobulin (IgM) exons M1 and M2 is directed by two juxtaposed regulatory elements, an enhancer and an inhibitor, located within the M2 exon. A primary function of the enhancer is to counteract the inhibitor, allowing splicing to occur. Here we show that the inhibitor contains two binding sites for polypyrimidine tract binding protein (PTB). Mutational analysis indicates that only one of these sites is necessary and sufficient to direct splicing inhibition both in vitro and in vivo. We demonstrate that the difference in activity of the two sites is explained by proximity to the intron. We further show that the presence of the enhancer results in the disruption of the PTB-inhibitor interaction, enabling splicing to occur. In the absence of the enhancer, splicing can be artificially activated by immuno-inhibition of PTB. Collectively, our results indicate that a single PTB binding site can function as an inhibitor that regulates alternative splicing both in vitro and in vivo.

Human papillomavirus 16 (HPV16) E6E7 pre-mRNA is bicistronic and has an intron in the E6 coding region with one 5′ splice site and two alternative 3′ splice sites, which produce E6∗I and E6∗II, respectively. If this intron remains unspliced, the resulting E6E7 mRNA expresses oncogenic E6. We found for the first time that the E6E7 pre-mRNA was efficiently spliced in vitro only when capped and that cellular cap-binding factors were involved in the splicing. The cap-dependent splicing of the E6E7 pre-mRNA was extremely efficient in cervical cancer-derived cells, producing mostly E6∗I, but inefficient in cells transfected with a common retrovirus expression vector, pLXSN16E6E7, due to the large size of this vector's exon 1. Further studies showed that efficient splicing of the E6E7 pre-mRNA depends on the distance of the cap-proximal intron from the RNA 5′ cap, with an optimal distance of less than 307 nt in order to facilitate better association of U1 small nuclear RNA with the intron 5′ splice site. The same was true for splicing of human β-globin RNA. Splicing of the E6E7 RNA provided more E7 RNA templates and promoted E7 translation, whereas a lack of RNA splicing produced a low level of E7 translation. Together, our data indicate that the distance between the RNA 5′ cap and cap-proximal intron is rate limiting for RNA splicing. HPV16 E6E7 pre-mRNA takes advantage of its small cap-proximal exon to confer efficient splicing for better E7 expression.

The general splicing factor U2AF65 recognizes the polypyrimidine tract (Py tract) that precedes 3′ splice sites and has three RNA recognition motifs (RRMs). The C-terminal RRM (RRM3), which is highly conserved, has been proposed to contribute to Py-tract binding and establish protein–protein contacts with splicing factors mBBP/SF1 and SAP155. Unexpectedly, we find that the human RRM3 domain is dispensable for U2AF65 activity in vitro. However, it has an essential function in Schizosaccharomyces pombe distinct from binding to the Py tract or to mBBP/SF1 and SAP155. First, deletion of RRM3 from the human protein has no effect on Py-tract binding. Second, RRM123 and RRM12 select similar sequences from a random pool of RNA. Third, deletion of RRM3 has no effect on the splicing activity of U2AF65 in vitro. However, deletion of the RRM3 domain of S. pombe U2AF59 abolishes U2AF function in vivo. In addition, certain amino acid substitutions on the four-stranded β-sheet surface of RRM3 compromise U2AF function in vivo without affecting binding to mBBP/SF1 or SAP155 in vitro. We propose that RRM3 has an unrecognized function that is possibly relevant for the splicing of only a subset of cellular introns. We discuss the implications of these observations on previous models of U2AF function.

Type 1 interferon can trigger flares of psoriasis. Hypersensitivity to type 1 interferon signaling causes a psoriasis-like skin disease in mice deficient for the transcription factor interferon regulatory factor 2 (IRF2). The human IRF2 gene is located at a previously identified candidate psoriasis susceptibility locus on chromosome 4q (PSORS3 at D4S1535). Therefore, we tested association of psoriasis with IRF2. We generated a sample consisting of 157 families with a total of 521 individuals. Five novel microsatellite markers were developed and typed, and complemented with three known markers to yield a set of eight markers spaced within 600 kb around the IRF2 gene, three of which are located in the gene. We detected association of IRF2 with type 1 psoriasis at two markers in the IRF2 gene. Haplotype sharing analysis confirmed association of IRF2 with type 1 psoriasis (p=0.0017; pcorr=0.03). The 921G/A SNP in exon 9 was found to obliterate a predicted exon splice enhancer in an allele-specific manner. There was a suggestive increase of homozygosity for the splicing-deficient allele in type 1 psoriasis patients. Our data identify IRF2 as a potential susceptibility gene for psoriasis.

In potato invertase genes, the constitutively included, 9-nucleotide (nt)-long mini-exon requires a strong branchpoint and U-rich polypyrimidine tract for inclusion. The strength of these splicing signals was demonstrated by greatly enhanced splicing of a poorly spliced intron and by their ability to support splicing of an artificial mini-exon, following their introduction. Plant introns also require a second splicing signal, UA-rich intronic elements, for efficient intron splicing. Mutation of the branchpoint caused loss of mini-exon inclusion without loss of splicing enhancement, showing that the same U-rich sequence can function as either a polypyrimidine tract or a UA-rich intronic element. The distinction between the splicing signals depended on intron context (the presence or absence of an upstream, adjacent and functional branchpoint), and on the sequence context of the U-rich elements. Polypyrimidine tracts tolerated C residues while UA-rich intronic elements tolerated As. Thus, in plant introns, U-rich splicing elements can have dual roles as either a general plant U-rich splicing signal or a polypyrimidine tract. Finally, overexpression of two different U-rich binding proteins enhanced intron recognition significantly. These results highlight the importance of co-operation between splicing signals, the importance of other nucleotides within U-rich elements for optimal binding of competing splicing factors and effects on splicing efficiency of U-rich binding proteins.

To improve transfection efficiency in Trypanosoma cruzi, we developed a new electroporation protocol and expression vectors which use luciferase and green and red fluorescent proteins as reporter genes. In transient transfections, the electroporation conditions reported here resulted in luciferase expression 100 times higher than the levels obtained with previously described protocols. To verify whether sequences containing different trans-splicing signals influence reporter gene expression, we compared DNA fragments corresponding to 5′ untranslated plus intergenic (5′ UTR plus Ig) regions from GAPDH, TcP2β, α- and β-tubulin and amastin genes. Vectors containing sequences derived from the first four genes presented similar efficiencies and resulted in luciferase expression in transiently transfected epimastigotes that was up to 10 times higher than that for a control vector. In contrast, the amastin 5′ UTR plus Ig resulted in lower levels of reporter gene expression. We also constructed a vector containing an expression cassette designed to be targeted to the tubulin locus of the parasite. Fil: DaRocha, Wanderson D.. Universidade Federal de Minas Gerais; Brasil Fil: Silva, Rosiane A.. Universidade Federal de Minas Gerais; Brasil Fil: Bartholomeu, Daniella C.. Universidade Federal de Minas Gerais; Brasil Fil: Pires, Simone F.. Universidade Federal de Minas Gerais; Brasil Fil: Freitas, Jorge M.. Universidade Federal de Minas Gerais; Brasil Fil: Macedo, Andrea M.. Universidade Federal de Minas Gerais; Brasil Fil: Vazquez, Martin Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires; Argentina Fil: Levin, Mariano Jorge. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires; Argentina Fil: Teixeira, Santuza M. R.. Universidade Federal de Minas Gerais; Brasil

Hotspot mutations in the spliceosome gene SF3B1 are reported in ∼20% of uveal melanomas. SF3B1 is involved in 3′-splice site (3′ss) recognition during RNA splicing; however, the molecular mechanisms of its mutation have remained unclear. Here we show, using RNA-Seq analyses of uveal melanoma, that the SF3B1R625/K666 mutation results in deregulated splicing at a subset of junctions, mostly by the use of alternative 3′ss. Modelling the differential junctions in SF3B1WT and SF3B1R625/K666 cell lines demonstrates that the deregulated splice pattern strictly depends on SF3B1 status and on the 3'ss-sequence context. SF3B1WT knockdown or overexpression do not reproduce the SF3B1R625/K666 splice pattern, qualifying SF3B1R625/K666 as change-of-function mutants. Mutagenesis of predicted branchpoints reveals that the SF3B1R625/K666-promoted splice pattern is a direct result of alternative branchpoint usage. Altogether, this study provides a better understanding of the mechanisms underlying splicing alterations induced by mutant SF3B1 in cancer, and reveals a role for alternative branchpoints in disease., Mutations in the splicing factor SF3B1 are found in uveal melanoma. Here, Alsafadi et al. use RNA-sequencing data from these cancers and experimental models, and show that mutant SF3B1 promotes alternative branchpoints in a specific gene subset and that the mutant protein gains a new function.

Vertebrate pre-mRNA transcripts contain many sequences that resemble splice sites on the basis of agreement to the consensus,yet these more numerous false splice sites are usually completely ignored by the cellular splicing machinery. Even at the level of exon definition,pseudo exons defined by such false splices sites outnumber real exons by an order of magnitude. We used a support vector machine to discover sequence information that could be used to distinguish real exons from pseudo exons. This machine learning tool led to the definition of potential branch points,an extended polypyrimidine tract,and C-rich and TG-rich motifs in a region limited to 50 nt upstream of constitutively spliced exons. C-rich sequences were also found in a region extending to 80 nt downstream of exons,along with G-triplet motifs. In addition,it was shown that combinations of three bases within the splice donor consensus sequence were more effective than consensus values in distinguishing real from pseudo splice sites; two-way base combinations were optimal for distinguishing 3' splice sites. These data also suggest that interactions between two or more of these elements may contribute to exon recognition,and provide candidate sequences for assessment as intronic splicing enhancers.

Transcription and pre-mRNA splicing are coordinated temporally and spatially, and both processes can influence each other. In particular, control of transcriptional elongation by RNA polymerase II has proved to be important for alternative splicing regulation. In this report we demonstrate that the efficiency of exon recognition by the splicing machinery is crucial for the elongation control. Alternative splicing of the fibronectin extra domain I (EDI) is because the polypyrimidine tract of its 3'-splice site occurs suboptimal. By mutating the polypyrimidine tract of EDI in two different positions, individually or in combination, and by disrupting its exonic splicing silencer, we managed to generate minigenes with increasing degrees of exon recognition. Improvement of exon recognition is evidenced by independence from the splicing regulator SF2/ASF for inclusion. The mutated minigenes were used to transfect human cells in culture and study the responsiveness of EDI alternative splicing to activation or inhibition of pol II elongation. Our results revealed that responsiveness of exon skipping to elongation is inversely proportional to 3'-splice site strength, which means that the better the alternative exon is recognized by the splicing machinery, the less its degree of inclusion is affected by transcriptional elongation.

Nonsyndromic inherited hearing impairment is genetically heterogeneous. Up to now, approximately 51 autosomal dominant loci implicated in nonsyndromic forms of hearing impairment have been reported in humans and 17 causative genes have been identified. Skipping of exon 8 in the DFNA5 gene has been shown to cause hearing impairment in a Dutch family. To our knowledge, no other DFNA5 mutation has been reported in familial or sporadic hearing impairment. Here, we report another mutation in DFNA5, a CTT deletion in the polypyrimidine tract of intron 7. This mutation, just like the previously reported mutation in the Dutch family, leads to skipping of exon 8 of DFNA5. In addition, we prove the existence of a recently identified short isoform of DFNA5, but the 3-nucleotide deletion reported here seems not to affect the function of this short isoform. Because no other mutation in any other part of DFNA5 has ever been described, this finding might indicate that exon 8 of DFNA5 is indispensable for the development of hearing impairment.

Knowledge of the cis-acting elements is required for identifying trans-acting splicing factors underlying cartilage-specific alternative splicing of Col2 pre-mRNA. By performing desired deletions in the mouse Col2 pre-mRNA, location of the intronic cis-acting elements was narrowed down to be at or near splice-junction sequences flanking exon 2 of the gene. Introduction: Type II collagen (Col2) pre-mRNA undergoes cartilage-specific alternative splicing involving exon 2 during chondrocyte differentiation. Thus, the trans-acting protein factors that regulate the splicing are associated with the differentiation of chondrocytes. Knowledge of the cognate cis-acting elements is necessary to eventually identify the trans-acting factors. Materials and Methods: To localize the cis-acting sequences, we created several deletions within a minigene containing exon 1 to exon 4 of mouse Col 2 gene and evaluated alternative splicing of the resulting pre-mRNAs in ATDC5 cells, a model of insulin-stimulated chondrocyte differentiation. The first deletion reduced intron 1 from 3799 to 259 bp, the second reduced intron 2 from 1108 to 94 bp, the third combined the above two deletions, and the fourth was derived from the third by removing intron 3 and exon 4. ATDC5 cells harboring these constructs were cultured for up to 21 days with or without insulin. Alternative splicing was evaluated by determining the ratio of Col2B (lacks exon 2) to Col2A (has exon 2) RNAs by reverse transcription-polymerase chain reaction. Results: The deletion in intron 1 had no effect on the alternative splicing while other deletions affected splicing (demonstrated by the presence of splicing intermediates) in cells cultured without insulin or with insulin for 1 week. The splicing intermediates were not seen from any construct when cells were cultured longer (14–21 days) with insulin. Conclusion: These results show that the 259-bp intron 1, the 94-bp intron 2, and exon 2 sequences retained in the fourth construct provide cis-acting signal sufficient for insulin-induced cartilage-specific alternative splicing of Col2 pre-mRNA.

Hepatitis C virus (HCV) translation is mediated by an IRES structure. Instead of a poly(A) tail, the 3' end of the genome contains a tripartite 3'NTR composed of a non-conserved region, a polypyrimidine tract and a highly conserved stretch of 98 nt, termed the 3'X region. Using a set of bicistronic recombinant DNA constructs expressing two reporter genes separated by the HCV IRES, it was determined whether the HCV 3'NTR sequence, in the presence or absence of HCV proteins, played a role in the efficiency of HCV IRES-dependent translation ex vivo. Bicistronic expression cassettes were transfected into hepatic and non-hepatic cell lines. These results show that neither the entire 3'NTR nor the 3'X sequence alters IRES-dependent translation efficiency, whatever the cell line tested. A potential effect of the 3'NTR on IRES-dependent translation in the presence of HCV proteins was investigated further. Neither non-structural nor structural HCV proteins had any effect on the efficiency of IRES in this system. In addition, in order to mimic HCV genome organization, monocistronic expression cassettes containing the IRES and a Core-DsRed fusion gene were constructed with or without the 3'NTR. In this context, no effect of the 3'NTR on IRES translation efficiency was observed, even in the presence of HCV proteins. These data demonstrate that HCV translation is not modulated by the viral genomic 3'NTR sequence, even in the presence of HCV structural or non-structural proteins.

Polypyrimidine tract-binding protein (PTB) is an abundant widespread RNA-binding protein with roles in regulation of pre-mRNA alternative splicing and 3'-end processing, internal ribosomal entry site-driven translation, and mRNA localization. Tissue-restricted paralogs of PTB have previously been reported in neuronal and hematopoietic cells. These proteins are thought to replace many general functions of PTB, but to have some distinct activities, e.g. in the tissue-specific regulation of some alternative splicing events. We report the identification and characterization of a fourth rodent PTB paralog (smPTB) that is expressed at high levels in a number of smooth muscle tissues. Recombinant smPTB localized to the nucleus, bound to RNA, and was able to regulate alternative splicing. We suggest that replacement of PTB by smPTB might be important in controlling some pre-mRNA alternative splicing events.

Isoforms of the smooth muscle myosin phosphatase targeting subunit 1 (MYPT1) are generated by cassette-type alternative splicing of exons. Tissue-specific expression of these isoforms is thought to determine smooth muscle-relaxant properties and unique responses to signaling pathways. We used mini-gene deletion/mutation constructs to identify cis regulators of splicing of the chicken MYPT1 central alternative exon. Comparisons of alternative exon splicing were made between smooth muscle cells of the fast-phasic contractile phenotype (gizzard), in which the central alternative exon is skipped, and slow tonic contractile phenotype (aorta), in which the alternative exon is included. We demonstrate that splicing of the alternative exon requires a cis-enhancer complex in the vicinity of the alternative exon 5'-splice site. This complex consists of two UCUU motifs in an intronic U-rich sequence (putative PTB (polypyrimidine tract binding) or T cell inhibitor of apoptosis-1 binding sites), an intronic 67-nucleotide enhancer that has similarities with the cardiac Troponin T MSE3 enhancer, and a potentially novel exonic splicing enhancer. The exonic enhancer contains the palindromic sequence UCCUACAUCCU present in many other transcripts where alternative splicing of exons occurs, suggesting that it may be more broadly active. The exonic enhancer is adjacent to a potentially novel exonic silencer element that contains a 13-nucleotide imperfect palindromic sequence. This silencer, in conjunction with a distal intronic silencer, is proposed to mediate the silencing of splicing of the MYPT1 central alternative exon in the fast phasic smooth muscle phenotype.

We describe the role of the Drosophila melanogaster hephaestus gene in wing development. We have identified several hephaestus mutations that map to a gene encoding a predicted RNA-binding protein highly related to human polypyrimidine tract binding protein and Xenopus laevis 60 kDa Vg1 mRNA-binding protein. Polypyrimidine tract binding proteins play diverse roles in RNA processing including the subcellular localization of mRNAs, translational control, internal ribosome entry site use, and the regulation of alternate exon selection. The analysis of gene expression in imaginal discs and adult cuticle of genetic mosaic animals supports a role for hephaestus in Notch signalling. Somatic clones lacking hephaestus express the Notch target genes wingless and cut , induce ectopic wing margin in adjacent wild-type tissue, inhibit wing-vein formation and have increased levels of Notch intracellular domain immunoreactivity. Clones mutant for both Delta and hephaestus have the characteristic loss-of-function thick vein phenotype of Delta . These results lead to the hypothesis that hephaestus is required to attenuate Notch activity following its activation by Delta. This is the first genetic analysis of polypyrimidine tract binding protein function in any organism and the first evidence that such proteins may be involved in the Notch signalling pathway.

Familial hypercholesterolemia (FH), a frequent monogenic condition complicated by premature cardiovascular disease, is characterized by high allelic heterogeneity at the low-density lipoprotein receptor (LDLR) locus. Despite more than a decade of genetic testing, knowledge about intronic disease-causing mutations has remained limited because of lack of available genomic sequences. Based on the finding from bioinformatic analysis that Alu repeats represent 85% of LDLR intronic sequences outside exon-intron junctions, we designed a strategy to improve the exploration of genomic regions in the vicinity of exons in 110 FH subjects from an admixed population. In the first group of 42 patients of negative mutation carriers, as previously established by former screening strategies (denaturing gradient gel electrophoresis, DNA sequencing with former primers overlapping splice-sites, Southern Blotting), about half (n=22) were found to be carriers of at least one heterozygous mutation. Among a second group of 68 newly recruited patients, 27% of mutation carriers (n=37) had a splicing regulatory mutation. Overall, out of the 54 mutations identified, 13 were intronic, and 18 were novel, out of which nearly half were intronic. Two novel intronic mutations (IVS8–10G→A within the polypyrimidine tract and IVS7+10G→A downstream of donor site) might create potential aberrant splice sites according to neural-network computed estimation, contrary to 31 common single nucleotide variations also identified at exon-intron junctions. This new strategy of detecting the most likely disease-causing LDLR mutations outside of Alu-rich genomic regions reveals that intronic mutations may have a greater impact than previously reported on the molecular basis of FH. Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00439-002-0813-4

The teschoviruses constitute a recently defined picornavirus genus. Most of the genome sequence of the porcine teschovirus-1 (PTV) Talfan and several other strains is known. We now demonstrate that initiation of protein synthesis occurs at nucleotide (nt) 412 on the PTV Talfan RNA and that nt 1 to 405 contains an internal ribosome entry site (IRES) that functions efficiently in vitro and within mammalian cells. In comparison with other picornavirus IRES elements, the PTV IRES is relatively short and lacks a significant polypyrimidine tract near the 3′ end. Expression of an enterovirus 2A protease, which induces cleavage of eIF4G within the translation initiation complex eIF4F, has little effect on the PTV IRES activity within BHK cells. The PTV IRES has a unique set of properties and represents a new class of picornavirus IRES element.

As an alternative to the scanning mechanism of initiation, the direct-internal-initiation mechanism postulates that the translational machinery assembles at the AUG start codon without traversing the entire 5' untranslated region (5'-UTR) of the mRNA. Although the existence of internal ribosome entry sites (IRESs) in viral mRNAs is considered to be well established, the existence of IRESs in cellular mRNAs has recently been challenged, in part because when testing is carried out using a conventional dicistronic vector, Northern blot analyses might not be sensitive enough to detect low levels of monocistronic transcripts derived via a cryptic promoter or splice site. To address this concern, we created a new promoterless dicistronic vector to test the putative IRES derived from the 5'-UTR of an mRNA that encodes the translation initiation factor eIF4G. Our analysis of this 5'-UTR sequence unexpectedly revealed a strong promoter. The activity of the internal promoter relies on the integrity of a polypyrimidine tract (PPT) sequence that had been identified as an essential component of the IRES. The PPT sequence overlaps with a binding site for transcription factor C/EBPbeta. Two other transcription factors, Sp1 and Ets, were also found to bind to and mediate expression from the promoter in the 5'-UTR of eIF4G mRNA. The biological significance of the internal promoter in the eIF4G mRNA might lie in the production of an N-terminally truncated form of the protein. Consistent with the idea that the cryptic promoter we identified underlies the previously reported IRES activity, we found no evidence of IRES function when a dicistronic mRNA containing the eIF4G sequence was translated in vitro or in vivo. Using the promoterless dicistronic vector, we also found promoter activities in the long 5'-UTRs of human Sno and mouse Bad mRNAs although monocistronic transcripts were not detectable on Northern blot analyses. The promoterless dicistronic vector might therefore prove useful in future studies to examine more rigorously the claim that there is IRES activity in cellular mRNAs.

The molecular basis of the skipping of constitutive exons in many messenger RNAs is not fully understood. A well-studied example is exon 9 of the human cystic fibrosis transmembrane conductance regulator gene (CFTR), in which an abbreviated polypyrimidine tract between the branch point A and the 3' splice site is associated with increased exon skipping and disease. However, many exons, both in CFTR and in other genes and have short polypyrimidine tracts in their 3' splice sites, yet they are not skipped. Inspection of the 5' splice sites immediately up- and downstream of exon 9 revealed deviations from consensus sequence, so we hypothesized that this exon may be inherently vulnerable to skipping. To test this idea, we constructed a CFTR minigene and replicated exon 9 skipping associated with the length of the polypyrimidine tract upstream of exon 9. We then mutated the flanking 5' splice sites and determined the effect on exon skipping. Conversion of the upstream 5' splice site to consensus by replacing a pyrimidine at position +3 with a purine resulted in increased exon skipping. In contrast, conversion of the downstream 5' splice site to consensus by insertion of an adenine at position +4 resulted in a substantial reduction in exon 9 skipping, regardless of whether the upstream 5' splice site was consensus or not. These results suggested that the native downstream 5' splice site plays an important role in CFTR exon 9 skipping, a hypothesis that was supported by data from sheep and mouse genomes. Although CFTR exon 9 in sheep is preceded by a long polypyrimidine tract (Y(14)), it skips exon 9 in vivo and has a nonconsensus downstream 5' splice site identical to that in humans. On the other hand, CFTR exon 9 in mice is preceded by a short polypyrimidine tract (Y(5)) but is not skipped in vivo. Its downstream 5' splice site differs from that in humans by a 2-nt insertion, which, when introduced into the human CFTR minigene, abolished exon 9 skipping. Taken together, these observations place renewed emphasis on deviations at 5' splice sites in nucleotides other than the invariant GT, particularly when such changes are found in conjunction with other altered splicing sequences, such as a shortened polypyrimidine tract. Thus, careful inspection of entire 5' splice sites may identify constitutive exons that are vulnerable to skipping.

The internal ribosome entry site (IRES) of picornaviruses has a 3' polypyrimidine tract (PPT) 16-24 bases upstream of an AUG triplet (PPT/AUG motif). This motif is critical in determining the efficiency of cap-independent translation. HAV has a conserved PPT/AUG motif consisting of a nine base sequence (AGGUUUUUC) 23 bases upstream of the preferred AUG start codon. This HAV-specific PPT/AUG motif is repeated and conserved in VP1 of HAV, but not of other picornaviruses. We proposed that the PPT/AUG motif in the open reading frame initiated translation and/or had an impact on the life cycle of the virus. In vitro translation of mutant bicistronic mRNAs and growth in cell culture of mutant viruses provided no evidence that the VP1 PPT/AUG motif had any impact on either translation or growth. HAV differs from other picornaviruses in its inefficient growth in cell culture. Since the HAV-specific PPT/AUG motif is found in only 1 in 300,000 reported viral sequences outside the hepatovirus genus, this motif may be a vestigial translation initiation element and may have played a role in determining the unusual phenotype of HAV.

The polypyrimidine tract-binding protein (PTB), an RNA-binding protein, is required for efficient translation of some mRNAs containing internal ribosomal entry sites (IRESs). Here we provide evidence that the addition of apoptosis-inducing agents to cells results in the cleavage of PTB isoforms 1, 2, and 4 by caspase-3. This cleavage of PTB separated the N-terminal region, containing NLS-RRM1, from the C-terminal region, containing RRM2-3-4. Our data indicate that there are three noncanonical caspase-3 target sites in PTBs, namely Ile-Val-Pro-Asp(7)Ile, Leu-Tyr-Thr-Asp(139)Ser, and Ala-Ala-Val-Asp(172)Ala. The C-terminal PTB fragments localized to the cytoplasm, as opposed to the nucleus where most intact PTBs are found. Moreover, these C-terminal PTB fragments inhibited translation of polioviral mRNA, which contains an IRES element requiring PTB for its activation. This suggests that translation of some IRES-containing mRNAs is regulated by proteolytic cleavage of PTB during apoptosis.

Summary Criteria for the identification of termination regions in Dictyostelium discoideum genes have been established and the sequence requirements for termination in 33 genes have been analyzed. A canonical hexamer signal AATAAA was present 15—30 nucleotides upstream of the cleavage site, usually a TA, and was embedded in a particularly A-rich environment. T- or GT-rich downstream elements characteristic of animal cells could not be identified. In a sample of 102 introns we have established the consensus AG/GTAAGT and ATAG/ for the 5′ and 3′ splice sites, respectively. Most introns are 75—150 nucleotides long and the A+T content is high (90%). A putative branch point was identified in half of the introns 20-60 nucleotides upstream of the 3′ splice site and the consensus TACTAAY was derived. A polypyrimidine tract required for branching in vertebrates was not identified, but weak preference for pyrimidine was found 10—45 nucleotides upstream of the 3′ splice site.

Mitochondrial ATP synthase gamma-subunit (F(1)gamma) pre-mRNA undergoes alternative splicing in a tissue- or cell type-specific manner. Exon 9 of F(1)gamma pre-mRNA is specifically excluded in heart and skeletal muscle tissues and in acid-stimulated human fibrosarcoma HT1080 cells, rhabdomyosarcoma KYM-1 cells, and mouse myoblast C2C12 cells. Recently, we found a purine-rich exonic splicing enhancer (ESE) element on exon 9 via transgenic mice bearing F(1)gamma mutant minigenes and demonstrated that this ESE functions ubiquitously with exception of muscle tissue (Ichida, M., Hakamata, Y., Hayakawa, M., Ueno E., Ikeda, U., Shimada, K., Hamamoto, T., Kagawa, Y., Endo, H. (2000) J. Biol. Chem. 275, 15992-16001). Here, we identified an exonic negative regulatory element responsible for muscle-specific exclusion of exon 9 using both in vitro and in vivo splicing systems. A supplementation assay with nuclear extracts from HeLa cells and acid-stimulated HT1080 cells was performed for an in vitro reaction of muscle-specific alternative splicing of F(1)gamma minigene and revealed that the splicing reaction between exons 8 and 9 was the key step for regulation of muscle-specific exon exclusion. Polypyrimidine tract in intron 8 requires ESE on exon 9 for constitutive splice site selection. Mutation analyses on the F(1)gammaEx8-9 minigene using a supplementation assay demonstrated that the muscle-specific negative regulatory element is positioned in the middle region of exon 9, immediately downstream from ESE. Detailed mutation analyses identified seven nucleotides (5'-AGUUCCA-3') as a negative regulatory element responsible for muscle-specific exon exclusion. This element was shown to cause exon skipping in in vivo splicing systems using acid-stimulated HT1080 cells after transient transfection of several mutant F(1)gammaEx8-9-10 minigenes. These results demonstrated that the 5'-AGUUCCA-3' immediately downstream from ESE is a muscle-specific exonic splicing silencer (MS-ESS) responsible for exclusion of exon 9 in vivo and in vitro.

The translation of polioviral mRNA occurs through an internal ribosomal entry site (IRES). Several RNA-binding proteins, such as polypyrimidine tract-binding protein (PTB) and poly(rC)-binding protein (PCBP), are required for the poliovirus IRES-dependent translation. Here we report that a poliovirus protein, 3C pro (and/or 3CD pro ), cleaves PTB isoforms (PTB1, PTB2, and PTB4). Three 3C pro target sites (one major target site and two minor target sites) exist in PTBs. PTB fragments generated by poliovirus infection are redistributed to the cytoplasm from the nucleus, where most of the intact PTBs are localized. Moreover, these PTB fragments inhibit polioviral IRES-dependent translation in a cell-based assay system. We speculate that the proteolytic cleavage of PTBs may contribute to the molecular switching from translation to replication of polioviral RNA.

The branchpoint sequence and associated polypyrimidine tract are firmly established splicing signals in vertebrates. In plants, however, these signals have not been characterized in detail. The potato invertase mini-exon 2 (9 nt) requires a branchpoint sequence positioned around 50 nt upstream of the 5' splice site of the neighboring intron and a U11 element found adjacent to the branchpoint in the upstream intron (Simpson et al., RNA, 2000, 6:422-433). Utilizing the sensitivity of this plant splicing system, these elements have been characterized by systematic mutation and analysis of the effect on inclusion of the mini-exon. Mutation of the branchpoint sequence in all possible positions demonstrated that branchpoints matching the consensus, CURAY, were most efficient at supporting splicing. Branchpoint sequences that differed from this consensus were still able to permit mini-exon inclusion but at greatly reduced levels. Mutation of the downstream U11 element suggested that it functioned as a polypyrimidine tract rather than a UA-rich element, common to plant introns. The minimum sequence requirement of the polypyrimidine tract for efficient splicing was two closely positioned groups of uridines 3-4 nt long (

Summary Mechanistic understanding of pre-mRNA splicing requires detailed structural information on various states of the spliceosome. Here we report the cryo electron microscopy (cryo-EM) structure of the human spliceosome just before exon ligation (the C ∗ complex) at an average resolution of 3.76 A. The splicing factor Prp17 stabilizes the active site conformation. The step II factor Slu7 adopts an extended conformation, binds Prp8 and Cwc22, and is poised for selection of the 3′-splice site. Remarkably, the intron lariat traverses through a positively charged central channel of RBM22; this unusual organization suggests mechanisms of intron recruitment, confinement, and release. The protein PRKRIP1 forms a 100-A α helix linking the distant U2 snRNP to the catalytic center. A 35-residue fragment of the ATPase/helicase Prp22 latches onto Prp8, and the quaternary exon junction complex (EJC) recognizes upstream 5′-exon sequences and associates with Cwc22 and the GTPase Snu114. These structural features reveal important mechanistic insights into exon ligation.