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PubMed1987

Minimal exon sequence requirements for efficient in vitro splicing of mono-intronic nuclear pre-mRNA.

Measurements of the in vitro splicing efficiency of deletion mutant RNA precursors containing the small intron of the rabbit beta-globin gene, which are truncated in the first or in the second exon, revealed that no more than approximately 20 nucleotides of either exon are necessary for efficient splicing. At least for the second exon, this minimal length requirement is globin sequence-independent. Reduction of the exon-2 length to 14 nucleotides resulted in very inefficient splicing, whereas further reduction to 5 nucleotides apparently abolished the second splicing step (3' cutting and ligation), whereas the first step (5' cutting and branching) still occurred. The splicing efficiency of a double-mutant substrate retaining approximately 20 nucleotides of each exon was reduced to 50%. A kinetic study indicated that in the reaction of this double-mutant substrate the second, but not the first, splicing step was delayed, in contrast to the reaction of the wild-type precursor. Duplication or triplication of the entire sequence of exon-1 did not affect the splicing efficiency, whereas elongation of this exon with approximately 100 nucleotides of 5'-flanking (nontranscribed) beta-globin sequence diminished the level of correct splicing with the simultaneous appearance of aberrant lariat forms. We conclude that for mono-intronic precursors in which there is only one choice of splice sites, most of the exon sequences are not mechanistically involved in the splicing process.

Animals↗
PubMed1995

SL1 trans-splicing specified by AU-rich synthetic RNA inserted at the 5' end of Caenorhabditis elegans pre-mRNA.

In Caenorhabditis elegans, pre-mRNAs of many genes are trans-spliced to one of two spliced leaders, SL1 or SL2. Some of those that receive exclusively SL1 have been characterized as having at their 5' ends outrons, AU-rich sequences similar to introns followed by conventional 3' splice sites. Comparison of outrons from many different SL1-specific C. elegans genes has not revealed the presence of any consensus sequence that might encode SL1-specificity. In order to determine what parameters influence the splicing of SL1, we performed in vivo experiments with synthetic splice sites. Synthetic AU-rich RNA, 51 nt or longer, placed upstream of a consensus 3' splice site resulted in efficient trans-splicing. With all sequences tested, this trans-splicing was specifically to SL1. Thus, no information beyond the presence of AU-rich RNA at least as long as the minimum-length C. elegans intron, followed by a 3' splice site, is required to specify trans-splicing or for strict SL1 specificity.

Adenine↗
PubMed1996

Functional analysis of an intron 3' splice site in Caenorhabditis elegans.

Caenorhabditis elegans introns typically lack both branch point and polypyrimidine tract consensus sequences utilized in other organisms for intron recognition. However, most introns have an unusually long, highly conserved consensus, UUUCAG/R, at the 3' splice site. This site can be recognized even when the -1G is changed to A. To determine how the 3' splice site is defined, we tested mutations in the sequence UUUCAA/A at the 3' splice site of the first intron of a test gene in vivo. Replacement of individual U's with A's or C's compromised splicing. When the sequence UUUCAA/AAG was tested, splicing occurred following both the -1A and the +3G, indicating that both UUUC and the AG contain 3' splice site information. When the sequence UUUCAA/AAA was tested, all splicing occurred following the -1A, suggesting that the UUUC contains sufficient information in the absence of an AG to specify the location of the splice site. In support of this idea, when point mutations were introduced into the UUUC, unspliced RNAs accumulated. Surprisingly, RNAs containing the mutant intron often contained the second, nonmutated intron as well, suggesting that interference with splicing of one intron can interfere with splicing of a second intron in the same pre-mRNA. The majority of these unspliced RNAs were degraded by the system responsible for degradation of transcripts containing nonsense mutations (smg), even though the intron contained no nonsense codon.

Animals↗
PubMed1996

Secondary structure of the yeast Saccharomyces cerevisiae pre-U3A snoRNA and its implication for splicing efficiency.

The Saccharomyces cerevisiae U3 snoRNA genes contain long spliceosomal introns with noncanonical branch site sequences. By using chemical and enzymatic methods to probe the RNA secondary structure and site-directed mutagenesis, we established the complete secondary structure of the U3A snoRNA precursor. This is the first determination of the complete secondary structure of an RNA spliced in a spliceosome. The peculiar cruciform structure of the U3A snoRNA 3'-terminal region is formed in the precursor RNA and the conserved Boxes B and C are accessible for binding the U3 snoRNP proteins. The intron forms a highly folded structure with a long central stem-loop structure that brings the 5' box and the branch site together. This is in agreement with the idea that secondary structure interactions are necessary for efficient splicing of long introns in yeast. The 3' splice site is in a bulged loop and the branch site sequence is single-stranded. Surprisingly, the 5' splice site is involved in a 6-base pair interaction. We used in vitro splicing experiments to show that, despite a noncanonical branch site sequence and a base paired 5' splice site, transcripts that mimic the authentic pre-U3A snoRNA are spliced very efficiently in vitro. Sequestering the 5' splice site in a more stable structure had a negative effect on splicing, which was partially compensated by converting the branch site sequence into a canonical sequence. Analysis of spliceosomal complex formation revealed a cumulative negative effect of a base pair interaction at the 5' splice site and of a deviation to the consensus sequence at the branch site on the efficiency of spliceosome formation in vitro.

Base Composition↗
PubMed1997

A highly stable duplex structure sequesters the 5' splice site region of hnRNP A1 alternative exon 7B.

Exon 7B in the hnRNP A1 pre-mRNA is alternatively spliced to yield A1 and A1(B), two proteins that differ in their ability to modulate 5' splice site selection. Sequencing the murine intron downstream of exon 7B revealed the existence of several regions of similarity to the corresponding human intron. In vitro splicing assays indicate that an 84-nt region (CE6IO) decreases splicing to the proximal 5' splice site in a pre-mRNA carrying the 5' splice sites of exon 7 and 7B. In vivo, the CE6IO element promotes exon 7B skipping in pre-mRNAs expressed from a mini-gene containing the hnRNP A1 alternative splicing unit. Using oligonucleotide-targeted RNase H cleavage assays, we provide support for the existence of highly stable base pairing interactions between CE6IO and the 5' splice site region of exon 7B. Duplex formation occurs in naked pre-mRNA, resists incubation in splicing extracts, and is associated with a reduction in the assembly of U1 snRNP-dependent complexes to the 5' splice site of exon 7B. Our results demonstrate that pre-mRNA secondary structure plays an important role in promoting exon 7B skipping in the A1 pre-mRNA.

Alternative Splicing↗
PubMed1997

Role of tissue specific alternative pre-mRNA splicing in the differentiation of the erythrocyte membrane.

Regulated alternative pre-mRNA splicing is neither as widely appreciated as a fundamental aspect of controlled gene expression nor as thoroughly studied as transcriptional regulation. However, as exemplified by the phenomena cited in this review, alternative splicing is a fundamentally important mechanism used in the eukaryotic world to enhance the range, versatility and plasticity of the structural information contained within a gene, and to create additional strategies by which the net quantitative output of a given gene product can be controlled. Regulation of RNA splicing gives genes a modularity that adds flexibility, and, therefore, selective advantage, to eukaryotes. It is likely, though unproven, that this opportunity for refined regulation and diversification provides at least one basis for the existence of the tandem exon-intron-exon structure found in the vast majority of eukaryotic genes and many viral genes. Many examples of alternative splicing are known, but, for the majority, no obvious biological impact of the alternatively spliced proteins on known cellular functions can be appreciated. Examples by which selectively regulated splicing pathways alter both the physiology and pathology of a major cellular event, such as differentiation and mechanical function of the red cell membrane, are thus relatively rare. The protein 4.1 gene and mRNA products thus provide an instructive and unusual system in which to explore the broader issue of the role of these regulatory mechanisms in the overall scheme of gene regulation and adaptation. The fact that hereditary hemolytic anemias result from mutations that directly or indirectly disrupt the splicing system emphasized the relevance of these mechanisms to molecular medicine. The features of splicing that we have reviewed in this paper, and the specific impact that regulated splicing exerts on differentiating red cells have, we hope, convinced the reader that RNA splicing is an important, fascinating, and potentially fruitful area for future study of human disease processes.

Actins↗
PubMed1993

Functional analysis of pre-mRNA splicing factor SF2/ASF structural domains.

Human pre-mRNA splicing factor SF2/ASF has an activity required for general splicing in vitro and promotes utilization of proximal alternative 5' splice sites in a concentration-dependent manner by opposing hnRNP A1. We introduced selected mutations in the N-terminal RNA recognition motif (RRM) and the C-terminal Arg/Ser (RS) domain of SF2/ASF, and assayed the resulting recombinant proteins for constitutive and alternative splicing in vitro and for binding to pre-mRNA and mRNA. Mutants inactive in constitutive splicing can affect alternative splice site selection, demonstrating that these activities involve distinct molecular interactions. Specific protein-RNA contact mediated by Phe56 and Phe58 in the RNP-1 submotif of the SF2/ASF RRM are essential for constitutive splicing, although they are not required for RRM-mediated binding to pre-mRNA. The RS domain is also required for constitutive splicing activity and both Arg and Ser residues are important. Analysis of domain deletion mutants demonstrated strong synergy between the RRM and a central degenerate RRM repeat in binding to RNA. These two domains are sufficient for alternative splicing activity in the absence of an RS domain.

Alternative Splicing↗
PubMed2026

The Role of Small Segmental Duplications in Generating Identical Isoforms Through Alternative Splicing Sites.

Alternative splicing plays a crucial role in expanding proteomic diversity but can also generate identical isoforms under certain conditions. While mutually exclusive splicing of tandem exons has occasionally been reported to produce identical isoforms, the extent to which other splicing events contribute to this phenomenon remains unclear. In this study, we demonstrate that alternative 5' and 3' splice site selection can also lead to the formation of identical isoforms, providing an additional type of splicing event for functional redundancy in transcriptomes. To address this, we analyzed reference genome annotations from 15 plant species, including Arabidopsis thaliana and wheat (Triticum aestivum), obtained from the RefSeq database. Identical isoforms were computationally defined as transcripts with distinct exon-intron structures but identical coding sequences. Our analysis reveals that the majority of alternative 5' and 3' fragments originate from small segmental duplications, suggesting that sequence repetition within gene regions facilitates the emergence of such splicing patterns. We also observed differences in the annotated 5' UTRs of some identical isoforms. However, since the alternative splicing sites themselves were not located within UTRs, these differences may reflect annotation uncertainty rather than genuine AS-derived variation. Given that UTR predictions in reference databases are not always precise, such observations should be interpreted cautiously. Expression analysis using an isoform-specific k-mer approach confirmed that identical isoforms can be differentially regulated. These findings suggest that, beyond expanding protein diversity, alternative splicing can also generate redundant isoforms that are differentially expressed at the RNA level, indicating potential regulatory roles. By elucidating the structural and regulatory factors contributing to the formation and retention of identical isoforms, our study provides new insights into the evolutionary and functional significance of alternative splicing in plants.

Alternative Splicing↗
PubMed2005

Characterization and prediction of alternative splice sites.

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 .

Alternative Splicing↗
PubMed2025

Utility of genome sequencing and group-enrichment to support splice variant interpretation in Marfan syndrome.

PURPOSE: To quantify the impact of noncanonical FBN1 splice site variants in undiagnosed Marfan syndrome (MFS), a connective tissue disorder associated with skeletal abnormalities and familial thoracic aortic aneurysm disease (FTAAD). METHODS: A systematic analysis of ultrarare FBN1 variants was performed using genome sequencing data from the 100,000 Genomes Project. Variants were annotated with SpliceAI and the significance of enrichment among individuals with FTAAD was assessed using Fisher's exact test. Experimental validation used RNA sequencing, reverse transcriptase polymerase chain reaction, minigene constructs, and replication analysis was with data from UK Biobank. RESULTS: Using aggregate data for 78,195 individuals, we identified 13,864 singleton single-nucleotide variants in FBN1 of which 21 were predicted to affect splicing (SpliceAI > 0.5). Incidence of candidate splice variants in individuals recruited with FTAAD (9/703) was significantly elevated compared with that seen in non-FTAAD participants (12/77,492; odds ratio = 84, P = 9.7 × 10-14). Additional analysis uncovered a further 14 families harboring 11 different FBN1 splice variants. A total of 20 candidate splice variants in 23 families were identified, of which 70% lay beyond the ±8 splice regions. RNA testing confirmed the predicted splice aberration in 16 of 20 and for 9 of 20, pseudoexonization was the likely splicing anomaly. CONCLUSION: Our findings indicate that noncanonical splice variants may account for approximately 3% of families with undiagnosed FTAAD, highlighting the importance of incorporating analysis of introns and confirmatory RNA testing into genetic testing for Marfan syndrome.

Humans↗
PubMed2005

Analysis of spliceosomal complexes in Trypanosoma brucei and silencing of two splicing factors Prp31 and Prp43.

In trypanosomatids all mRNAs undergo trans-splicing, whereas cis-splicing is restricted to a few transcripts. Trans-splicing is mechanistically similar to cis-splicing, however, little is known about the trans-splicing machinery and its underlying mechanism. In this study, we examined the involvement of splicing factors in cis- and trans-splicing by RNA interference (RNAi). Two factors (Prp31 and Prp43) were found to be essential for both pathways, suggesting that splicing factors are shared by these two reactions. We identified a 45S complex carrying pre-mRNA and all the U-snRNAs, including U1 and the SL RNA, suggesting that a single spliceosomal complex may potentially conduct both trans- and cis-splicing.

Animals↗
PubMed2005

Substrate-dependent differences in U2AF requirement for splicing in adenovirus-infected cell extracts.

U2AF has been characterized as an essential splicing factor required for efficient recruitment of U2 small nuclear ribonucleoprotein to the 3'-splice site in a pre-mRNA. The U2AF65 subunit binds to the pyrimidine tract of the pre-mRNA, whereas the U2AF(35) subunit contacts the 3'-splice site AG. Here we show that U2AF35 appears to be completely dispensable for splicing in nuclear extracts prepared from adenovirus late-infected cells (Ad-NE). As a consequence, the viral IIIa and cellular IgM introns, which both have suboptimal 3'-splice sites and require U2AF35 for splicing in nuclear extracts from uninfected cells, are transformed to U2AF35-independent introns in Ad-NE. Furthermore, we present evidence that two parallel pathways of 3'-splice site recognition exist in Ad-NE. We show that the viral 52,55K intron, which has an extended pyrimidine tract, requires U2AF for activity in Ad-NE. In contrast, the IgM intron, which has a weak 3'-splice site sequence context, undergoes the first catalytic step of splicing in U2AF-depleted Ad-NE, suggesting that spliceosome assembly occurs through a novel U2AF-independent pathway in Ad-NE.

Adenoviridae↗
PubMed1998

Genetic analysis of the SR protein ASF/SF2: interchangeability of RS domains and negative control of splicing.

The SR proteins constitute a family of splicing factors, highly conserved in metazoans, that contain one or two amino-terminal RNA-binding domains (RBDs) and a region enriched in arginine/serine repeats (RS domain) at the carboxyl terminus. Previous studies have shown that SR proteins possess distinct RNA-binding specificities that likely contribute to their unique functions, but it is unclear whether RS domains have specific roles in vivo. Here, we used a genetic system developed in the chicken B cell line DT40 to address this question. Expression of chimeric proteins generated by fusion of the RS domains of heterologous SR proteins, or a human TRA-2 protein, with the RBDs of ASF/SF2 allowed cell growth following genetic inactivation of endogenous ASF/SF2, indicating that RS domains are interchangeable for all functions required to maintain cell viability. However, a chimera containing the RS domain from a related splicing factor, U2AF65, could not rescue viability and was inactive in in vitro splicing assays, suggesting that this domain performs a distinct function. We also used the DT40 system to show that depletion of ASF/SF2 affects splicing of specific transcripts in vivo. Although splicing of several simple constitutive introns was not significantly affected, the alternative splicing patterns of two model pre-mRNAs switched in a manner consistent with predictions from previous studies. Unexpectedly, ASF/SF2 depletion resulted in a substantial increase in splicing of an HIV-1 tat pre-mRNA substrate, indicating that ASF/SF2 can repress tat splicing in vivo. These results provide the first demonstration that an SR protein can influence splicing of specific pre-mRNAs in vivo.

Alternative Splicing↗
PubMed2003

Impact of alternative initiation, splicing, and termination on the diversity of the mRNA transcripts encoded by the mouse transcriptome.

We analyzed the FANTOM2 clone set of 60,770 RIKEN full-length mouse cDNA sequences and 44,122 public mRNA sequences. We developed a new computational procedure to identify and classify the forms of splice variation evident in this data set and organized the results into a publicly accessible database that can be used for future expression array construction, structural genomics, and analyses of the mechanism and regulation of alternative splicing. Statistical analysis shows that at least 41% and possibly as much as 60% of multiexon genes in mouse have multiple splice forms. Of the transcription units with multiple splice forms, 49% contain transcripts in which the apparent use of an alternative transcription start (stop) is accompanied by alternative splicing of the initial (terminal) exon. This implies that alternative transcription may frequently induce alternative splicing. The fact that 73% of all exons with splice variation fall within the annotated coding region indicates that most splice variation is likely to affect the protein form. Finally, we compared the set of constitutive (present in all transcripts) exons with the set of cryptic (present only in some transcripts) exons and found statistically significant differences in their length distributions, the nucleotide distributions around their splice junctions, and the frequencies of occurrence of several short sequence motifs.

Alternative Splicing↗
PubMed2006

In vivo requirement of the small subunit of U2AF for recognition of a weak 3' splice site.

The U2 snRNP auxiliary factor (U2AF) is an essential splicing factor composed of two subunits, a large, 65-kDa subunit (U2AF(65)) and a small subunit, U2AF(35). U2AF(65) binds to the polypyrimidine tract upstream from the 3' splice site and promotes U2 snRNP binding to the pre-mRNA. Based on in vitro studies, it has been proposed that U2AF(35) plays a role in assisting U2AF(65) recruitment to nonconsensus polypyrimidine tracts. Here we have analyzed in vivo the roles of the two subunits of U2AF in the selection between alternative 3' splice sites associated with polypyrimidine tracts of different strengths. Our results reveal a feedback mechanism by which RNA interference (RNAi)-mediated depletion of U2AF(65) triggers the downregulation of U2AF(35). We further show that the knockdown of each U2AF subunit inhibits weak 3' splice site recognition, while overexpression of U2AF(65) alone is sufficient to activate the selection of this splice site. A variant of U2AF(65) lacking the interaction domain with U2AF(35) shows a reduced ability to promote this splicing event, suggesting that recognition of the weak 3' splice site involves the U2AF heterodimer. Furthermore, our data suggest that, rather than being required for splicing of all pre-mRNA substrates containing a weak polypyrimidine tract, U2AF(35) regulates the selection of weak 3' splice sites in a specific subset of cellular transcripts.

Base Sequence↗
PubMed1995

Intrinsic U2AF binding is modulated by exon enhancer signals in parallel with changes in splicing activity.

A functional analysis of exon replacement mutations was performed in parallel with RNA-protein binding assays to gain insight into the role of the exon in alternative and simple splicing events. These results show that constitutive exons from unrelated genes contain strong signals that promote splicing in multiple sequence contexts by enhancing 3' splice site activity. A clue to the nature of the relationship between the exon and adjacent 3' splice site is indicated by the binding properties of exon variant RNAs when tested with different biochemical preparations of the essential splicing protein, U2AF. In the context of a complete nuclear extract, U2AF binding to the 3' splice site is stimulated by the presence of an adjacent constitutive exon. In contrast, highly purified HeLa U2AF binds equivalently to the exon variants under conditions in which differential polypyrimidine tract binding is evident. These results provide support for an assisted binding model in which positive-acting signals within exons, exon enhancers, direct the binding of accessory factors, which in turn increase the intrinsic affinity of U2AF for the adjacent 3' splice site. Further support for an assisted binding model is indicated by biochemical complementation of U2AF binding and by the localization of a novel exon enhancer, which, when introduced into a weak exon, stimulates splicing activity in parallel with U2AF binding. Immunoprecipitation analysis identifies the splicing factor, SC35, as a constituent of the exon enhancer binding complex. These results are discussed in the context of current models for functional exon-bridging interactions.

Base Sequence↗
PubMed1993

Protein splicing of the yeast TFP1 intervening protein sequence: a model for self-excision.

Protein splicing is the protein analogue of RNA splicing in which the central portion (spacer) of a protein precursor is excised and the amino- and carboxy-terminal portions of the precursor reconnected. The yeast Tfp1 protein undergoes a rapid protein splicing reaction to yield a spliced 69 kDa polypeptide and an excised 50 kDa spacer protein. We have demonstrated that the 69 kDa species arises by reformation of a bona fide peptide bond. Deletion analyses indicate that only sequences in the central spacer protein of the Tfp1 precursor are critical for the protein splicing reaction. A fusion protein in which only the Tfp1 spacer domain was inserted into an unrelated protein also underwent efficient splicing, demonstrating that all of the information required for protein splicing resides within the spacer domain. Alteration of Tfp1p splice junction residues blocked or kinetically impaired protein splicing. A protein splicing model is presented in which asparagine rearrangement initiates the self-excision of the spacer protein from the Tfp1 precursor. The Tfp1 spacer protein belongs to a new class of intervening sequences that are excised at the protein rather than the RNA level.

Amino Acid Sequence↗
PubMed1983

Human beta-globin pre-mRNA synthesized in vitro is accurately spliced in Xenopus oocyte nuclei.

To study the mechanisms of RNA splicing we have synthesized beta-globin mRNA precursors by in vitro transcription using a plasmid in which a human beta-globin gene is fused to an efficient bacteriophage promoter. The structural requirements for accurate splicing of the in vitro synthesized pre-mRNAs were investigated by injection into Xenopus oocyte nuclei. We detect splicing only if the pre-mRNA is capped in vitro prior to injection; uncapped RNA is rapidly degraded. In addition, we find that in vitro synthesized pre-mRNAs that are not polyadenylated or lack a normal 3' end are spliced following injection into oocytes. The observation that a purified pre-mRNA can be spliced in oocytes indicates that transcription and splicing are not obligatorily coupled. When an in vitro synthesized beta-globin pre-mRNA containing a splice junction mutation is microinjected, the affected junction is not spliced, indicating that sequences necessary for accurate splicing in human cells are also necessary for splicing in oocytes.

Animals↗