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At least 1,135 records · Page 63Linked to original sources
PubMed1998

Modulation of protein splicing of the Saccharomyces cerevisiae vacuolar membrane ATPase intein.

Protein splicing of the Saccharomyces cerevisiae vacuolar membrane ATPase intein involves four highly coordinated reactions that result in precise cleavage and formation of peptide bonds. In this study, we investigated the roles of the last N-extein residue (-1 residue) and the intein penultimate residue in modulating splicing reactions. Most of the 20 amino acid substitutions at the -1 position had no effect on overall protein splicing but could lead to significant accumulation of thioester intermediates when splicing was blocked by mutation. A subset of -1 substitutions attenuated the initiation of protein splicing and enabled us to demonstrate in vitro splicing of a mesophilic intein containing all wild-type catalytic residues. Substitutions involving the intein penultimate residue allowed modulation of the branch resolution and C-terminal cleavage reaction. Our data suggest that the N-S acyl rearrangement, which initiates splicing, may also serve as the rate-limiting step. Through appropriate amino acid substitutions, we were able to modulate splicing reactions in vitro by change in pH or temperature or addition of thiol reagents. Both insertion and deletion were tolerated in the central region of the intein although splicing or structure of the intein may have been affected.

Amino Acid Substitution↗
PubMed2001

Dimethyl sulfoxide affects the selection of splice sites.

Depending on the cell lines and cell types, dimethyl sulfoxide (Me2SO) can induce or block cell differentiation and apoptosis. Although Me2SO treatment alters many levels of gene expression, the molecular processes that are directly affected by Me2SO have not been clearly identified. Here, we report that Me2SO affects splice site selection on model pre-mRNAs incubated in a nuclear extract prepared from HeLa cells. A shift toward the proximal pair of splice sites was observed on pre-mRNAs carrying competing 5'-splice sites or competing 3'-splice sites. Because the activity of recombinant hnRNP A1 protein was similar when added to extracts containing or lacking Me2SO, the activity of endogenous A1 proteins is probably not affected by Me2SO. Notably, in a manner reminiscent of SR proteins, Me2SO activated splicing in a HeLa S100 extract. Moreover, the activity of recombinant SR proteins in splice site selection in vitro was improved by Me2SO. Polar solvents like DMF and formamide similarly modulated splice site selection in vitro but formamide did not activate a HeLa S100 extract. We propose that Me2SO improves ionic interactions between splicing factors that contain RS-domains. The direct impact of Me2SO on alternative splicing may explain, at least in part, the different and sometimes opposite effects of Me2SO on cell differentiation and apoptosis.

3' Untranslated Regions↗
PubMed2004

Manipulation of alternative splicing by a newly developed inhibitor of Clks.

The regulation of splice site usage provides a versatile mechanism for controlling gene expression and for the generation of proteome diversity, playing an essential role in many biological processes. The importance of alternative splicing is further illustrated by the increasing number of human diseases that have been attributed to mis-splicing events. Appropriate spatial and temporal generation of splicing variants demands that alternative splicing be subjected to extensive regulation, similar to transcriptional control. The Clk (Cdc2-like kinase) family has been implicated in splicing control and consists of at least four members. Through extensive screening of a chemical library, we found that a benzothiazole compound, TG003, had a potent inhibitory effect on the activity of Clk1/Sty. TG003 inhibited SF2/ASF-dependent splicing of beta-globin pre-mRNA in vitro by suppression of Clk-mediated phosphorylation. This drug also suppressed serine/arginine-rich protein phosphorylation, dissociation of nuclear speckles, and Clk1/Sty-dependent alternative splicing in mammalian cells. Consistently, administration of TG003 rescued the embryonic defects induced by excessive Clk activity in Xenopus. Thus, TG003, a novel inhibitor of Clk family will be a valuable tool to dissect the regulatory mechanisms involving serine/arginine-rich protein phosphorylation signaling pathways in vivo, and may be applicable for the therapeutic manipulation of abnormal splicing.

Alternative Splicing↗
PubMed2006

Fox-2 splicing factor binds to a conserved intron motif to promote inclusion of protein 4.1R alternative exon 16.

Activation of protein 4.1R exon 16 (E16) inclusion during erythropoiesis represents a physiologically important splicing switch that increases 4.1R affinity for spectrin and actin. Previous studies showed that negative regulation of E16 splicing is mediated by the binding of heterogeneous nuclear ribonucleoprotein (hnRNP) A/B proteins to silencer elements in the exon and that down-regulation of hnRNP A/B proteins in erythroblasts leads to activation of E16 inclusion. This article demonstrates that positive regulation of E16 splicing can be mediated by Fox-2 or Fox-1, two closely related splicing factors that possess identical RNA recognition motifs. SELEX experiments with human Fox-1 revealed highly selective binding to the hexamer UGCAUG. Both Fox-1 and Fox-2 were able to bind the conserved UGCAUG elements in the proximal intron downstream of E16, and both could activate E16 splicing in HeLa cell co-transfection assays in a UGCAUG-dependent manner. Conversely, knockdown of Fox-2 expression, achieved with two different siRNA sequences resulted in decreased E16 splicing. Moreover, immunoblot experiments demonstrate mouse erythroblasts express Fox-2. These findings suggest that Fox-2 is a physiological activator of E16 splicing in differentiating erythroid cells in vivo. Recent experiments show that UGCAUG is present in the proximal intron sequence of many tissue-specific alternative exons, and we propose that the Fox family of splicing enhancers plays an important role in alternative splicing switches during differentiation in metazoan organisms.

Amino Acid Motifs↗
PubMed2000

Determinants of 4-repeat tau expression. Coordination between enhancing and inhibitory splicing sequences for exon 10 inclusion.

Mutations in the tau gene are pathogenic causing autosomal dominant frontotemporal dementia with Parkinsonism-chromosome 17 type (FTDP-17). Some mutations in tau exon 10 (E10) and immediately adjacent sequences cause disease by altering E10 splicing. To determine the mechanism of normal E10 splicing regulation and how FTDP-17 mutations alter splicing, mutational analysis of E10 was performed. The results show that E10 contains a complex array of both enhancer and inhibitor cis-acting elements that modulate usage of a weak 5' splice site. The 5' end of E10 contains a previously unrecognized multipartite exon splicing enhancer (ESE) composed of an SC35-like binding sequence, a purine-rich sequence, and an AC-rich element. Downstream of this ESE is a purine-rich exon splicing inhibitor. Intronic sequences immediately downstream of E10 also are inhibitory. The results support an alternative model in which I10 inhibitory sequences appear to function as a linear sequence. The cis-elements described are not redundant, and all appear required for normal E10 splicing. Results with double mutations demonstrate that the ESE and the intronic inhibitory element collaborate to regulate splicing. Thus splicing of tau E10 is regulated by a complex set of cis-acting elements that span nearly the entire exon and also include intronic sequences.

5' Untranslated Regions↗
PubMed2003

Alterations of mRNA splicing in primary effusion lymphomas.

Primary effusion lymphoma (PEL) is a unique form of malignant lymphoma associated with infection by the Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus-8 (HHV-8). The majority of PELs also contain the EBV genome. Although viral infection is believed to play a critical role in the pathogenesis of PEL, it has been suggested that additional molecular lesions are required for the development of PEL. Alternative splicing of pre-mRNA is an important mechanism in the regulation of cellular and viral gene expression. Deregulation of pre-mRNA splicing may shift the gene expression balance and lead to the development of cancer. In order to investigate mRNA splicing in PELs, we examined mRNA splicing of three genes, DNA polymerase beta (pol beta), Bcl-x and CD45, in eight PEL cell lines. We found that the average variant percentage of pol beta in PEL cell lines is two times higher than in peripheral blood mononuclear cells (PBMC) and that the variant pattern of genes bcl-x and CD45 is quite different in PEL cell lines than in PBMC. In addition, we also found that the percentage of variant pol beta increased two-fold in PBMC following Epstein-Barr virus (EBV) infection. Therefore, viral infection may contribute to mRNA alternative splicing in PEL. In order to explore the mechanism by which viral infection affects mRNA splicing, we also examined the roles of genes KS-SM, SM and EBERs and viral copies in mRNA splicing. Our findings indicate that various factors acting as positive or negative regulators may be involved in mRNA alternative splicing caused by viral infection. In conclusion, mRNA splicing in PEL can be altered by viral infection and this alteration may contribute to the pathogenesis of PEL.

Cell Transformation, Viral↗
PubMed1997

A functional interaction between the carboxy-terminal domain of RNA polymerase II and pre-mRNA splicing.

In the preceding study we found that Sm snRNPs and SerArg (SR) family proteins co-immunoprecipitate with Pol II molecules containing a hyperphosphorylated CTD (Kim et al., 1997). The association between Pol IIo and splicing factors is maintained in the absence of pre-mRNA, and the polymerase need not be transcriptionally engaged (Kim et al., 1997). The latter findings led us to hypothesize that a phosphorylated form of the CTD interacts with pre-mRNA splicing components in vivo. To test this idea, a nested set of CTD-derived proteins was assayed for the ability to alter the nuclear distribution of splicing factors, and to interfere with splicing in vivo. Proteins containing heptapeptides 1-52 (CTD52), 1-32 (CTD32), 1-26 (CTD26), 1-13 (CTD13), 1-6 (CTD6), 1-3 (CTD3), or 1 (CTD1) were expressed in mammalian cells. The CTD-derived proteins become phosphorylated in vivo, and accumulate in the nucleus even though they lack a conventional nuclear localization signal. CTD52 induces a selective reorganization of splicing factors from discrete nuclear domains to the diffuse nucleoplasm, and significantly, it blocks the accumulation of spliced, but not unspliced, human beta-globin transcripts. The extent of splicing factor disruption, and the degree of inhibition of splicing, are proportional to the number of heptapeptides added to the protein. The above results indicate a functional interaction between Pol II's CTD and pre-mRNA splicing.

Amino Acid Sequence↗
PubMed1999

Mechanism of non-spliceosomal mRNA splicing in the unfolded protein response pathway.

The unfolded protein response is an intracellular signaling pathway that, in response to accumulation of misfolded proteins in the lumen of the endoplasmic reticulum (ER), upregulates transcription of ER resident chaperones. A key step in this pathway is the non-conventional, regulated splicing of the mRNA encoding the positive transcriptional regulator Hac1p. In the yeast Saccharomyces cerevisiae, the bifunctional transmembrane kinase/endoribonuclease Ire1p cleaves HAC1 mRNA at both splice junctions and tRNA ligase joins the two exons together. We have reconstituted HAC1 mRNA splicing in an efficient in vitro reaction and show that, in many ways, the mechanism of HAC1 mRNA splicing resembles that of pre-tRNA splicing. In particular, Ire1p endonucleolytic cleavage leaves 2', 3'-cyclic phosphates, the excised exons remain associated by base pairing, and exon ligation by tRNA ligase follows the same chemical steps as for pre-tRNA splicing. To date, this mechanism of RNA processing is unprecedented for a messenger RNA. In contrast to the striking similarities to tRNA splicing, the structural features of the splice junctions recognized by Ire1p differ from those recognized by tRNA endonuclease. We show that small stem-loop structures predicted to form at both splice junctions of HAC1 mRNA are required and sufficient for Ire1p cleavage.

Base Pairing↗
PubMed2003

Efficient use of a 'dead-end' GA 5' splice site in the human fibroblast growth factor receptor genes.

We have investigated use of a conserved non-canonical GA 5' splice site present in vertebrate fibroblast growth factor receptor (FGFR) genes. Despite previous studies suggesting that GA at the beginning of an intron is incompatible with splicing, we observe efficient utilization of this splice site for human FGFR1 gene constructs. We show that use of the GA splice site is dependent on both a conventional splice site six nucleotides upstream and sequence elements within the downstream intron. Furthermore, our results are consistent with competition between the tandem 5' splice sites being mediated by U6 snRNP, rather than U1 snRNP. Thus the GA 5' splice site represents an extension of the adjacent conventional 5' splice site, the first natural example of such a composite 5' splice site.

Alternative Splicing↗
PubMed1999

The role of evolutionarily conserved sequences in alternative splicing at the 3' end of Drosophila melanogaster myosin heavy chain RNA.

Exon 18 of the muscle myosin heavy chain gene (Mhc) of Drosophila melanogaster is excluded from larval transcripts but included in most adult transcripts. To identify cis-acting elements regulating this alternative RNA splicing, we sequenced the 3' end of Mhc from the distantly related species D. virilis. Three noncoding regions are conserved: (1) the nonconsensus splice junctions at either end of exon 18; (2) exon 18 itself; and (3) a 30-nucleotide, pyrimidine-rich sequence located about 40 nt upstream of the 3' splice site of exon 18. We generated transgenic flies expressing Mhc mini-genes designed to test the function of these regions. Improvement of both splice sites of adult-specific exon 18 toward the consensus sequence switches the splicing pattern to include exon 18 in all larval transcripts. Thus nonconsensus splice junctions are critical to stage-specific exclusion of this exon. Deletion of nearly all of exon 18 does not affect stage-specific utilization. However, splicing of transcripts lacking the conserved pyrimidine sequence is severely disrupted in adults. Disruption is not rescued by insertion of a different polypyrimidine tract, suggesting that the conserved pyrimidine-rich sequence interacts with tissue-specific splicing factors to activate utilization of the poor splice sites of exon 18 in adult muscle.

Alternative Splicing↗
PubMed2005

Human-mouse comparative analysis reveals that branch-site plasticity contributes to splicing regulation.

The formation of base-pairing between the branch-site (BS) sequence and the U2 snRNP is an important step in mRNA splicing. We developed a new algorithm to identify both the BS sequence and the polypyrimidine tract (PPT) and validated its predictions experimentally. To assess BS conservation between human and mouse, we assembled and analyzed 46 812 and 242 constitutively and alternatively spliced orthologs of human-mouse intron pairs, respectively. Combinations of BSs and PPTs can be found in most of the constitutive and alternative introns. The average distance between the BS and the 3' splice site (3'ss) is 33-34 nt. Acceptor-like AG dinucleotides that resided between the predicted BS and the 3'ss were found to appear mostly within 5 nt, but not more than 19 nt, downstream of the BS. However, although 32% of homologous alternatively spliced BS sequences were fully conserved between human and mouse, only a small fraction (3%) of homologous constitutive counterparts was fully conserved. This indicates that the full sequence of the BS is under weak purifying selection in constitutively spliced introns and further strengthens the view that the BS sequence is just one of several factors determining the ability of the splicing machinery to identify the BS location. Mutations in the putative BS revealed a shift from constitutive to alternative splicing, and it also controls the inclusion/skipping ratio in alternative splicing. This suggests a role for BS sequences in regulated splicing.

Algorithms↗
PubMed2005

Changes in alternative splicing of human and mouse genes are accompanied by faster evolution of constitutive exons.

Alternative splicing is known to be an important source of protein sequence variation, but its evolutionary impact has not been explored in detail. Studying alternative splicing requires extensive sampling of the transcriptome, but new data sets based on expressed sequence tags aligned to chromosomes make it possible to study alternative splicing on a genome-wide scale. Although genes showing alternative splicing by exon skipping are conserved as compared to the genome as a whole, we find that genes where structural differences between human and mouse result in genome-specific alternatively spliced exons in one species show almost 60% greater nonsynonymous divergence in constitutive exons than genes where exon skipping is conserved. This effect is also seen for genes showing species-specific patterns of alternative splicing where gene structure is conserved. Our observations are not attributable to an inherent difference in rate of evolution between these two sets of proteins or to differences with respect to predictors of evolutionary rate such as expression level, tissue specificity, or genetic redundancy. Where genome-specific alternatively spliced exons are seen in mammals, the vast majority of skipped exons appear to be recent additions to gene structures. Furthermore, among genes with genome-specific alternatively spliced exons, the degree of nonsynonymous divergence in constitutive sequence is a function of the frequency of incorporation of these alternative exons into transcripts. These results suggest that alterations in alternative splicing pattern can have knock-on effects in terms of accelerated sequence evolution in constant regions of the protein.

Alternative Splicing↗
PubMed1985

Intron splicing: a conserved internal signal in introns of Drosophila pre-mRNAs.

The introns of Drosophila pre-mRNAs have been analysed for conserved internal sequence elements near the 3' intron boundary similar to the T-A-C-T-A-A-C in yeast introns and the C/T-T-A/G-A-C/T in introns of other organisms. Such conserved internal elements are the 3' splice signals recognized in intron splicing. In the lariat splicing mechanism, the G at the 5' end of an intron joins covalently to the last A of a 3' splice signal to form a branch point in a splicing intermediate. Analysis of 39 published sequences of Drosophila introns reveals that potential 3' splice signals with the consensus C/T-T-A/G-A-C/T are present in 18 cases. In 17 of the remaining cases signals are present which vary from this consensus just in the middle or last position. In Drosophila introns the 3' splice signal is usually located in a discrete region between 18 and 35 nucleotides upstream from the 3' splice point. We note that the Drosophila small nuclear U2-RNA has sequences complementary to C-T-G-A-T, one variant of the signal, and to C-A-G, one variant of the 3' terminus of an intron. We also note that the absence of any A-G between -3 and -19 from the 3' splice point may be an essential feature of a strong 3' boundary.

Animals↗
PubMed1986

Alternative splicing of SV40 early pre-mRNA in vitro.

Simian virus 40 (SV40) early pre-mRNA is spliced using either of two alternative 5' splice sites and a common 3' splice site to produce two mRNAs that encode the T and t antigens. We have studied alternative splicing of SV40 early pre-mRNA in vitro using a HeLa cell nuclear extract. Synthetic SV40 early transcripts are processed to T and t antigen mRNAs in vitro. As in SV40-infected cells in vivo, cleavage at the T antigen 5' splice site is more efficient than cleavage at the t antigen 5' splice site in vitro, although both of these 5' splice sites are utilized relatively inefficiently in vitro. The ratio of cleavage at the T and t antigen 5' splice sites is not changed significantly by a number of alterations in the conditions under which the in vitro splicing reactions are carried out.

Animals↗
PubMed1991

Splice site requirement for the efficient accumulation of polyoma virus late mRNAs.

Polyoma virus late nuclear primary transcripts are giant and heterogeneous, containing tandem repeats of the late strand of the circular viral genome. Late pre-mRNA processing involves the splicing of noncoding 'leader' exons to each other (removing genome-length introns), with the joining of the last leader to a coding 'body' exon. We have constructed a number of mutants blocked only in leader-leader splicing, or blocked in both leader-leader and leader-body splicing. We examined the accumulation of both nuclear and cytoplasmic late-strand RNAs in NIH3T3 cells. Consistent with our previous results, mutants lacking the 3' splice site of the late leader (leader-leader splicing blocked) showed a 10-20 fold defect in late RNA accumulation. Mutants which lacked the leader 5' splice site (leader-body splicing blocked) had a more profound defect, exhibiting virtually no late-strand cytoplasmic or nuclear RNA. This result was unexpected as a substantial proportion of wild type late cytoplasmic messages are unspliced. A mutant with no intron, but having functional 3' and 5' splice sites bordering the leader exon, is capable of producing large amounts of unspliced late mRNA. This demonstrates that an excisable intron is not a requirement for late mRNA accumulation. The accumulation of polyoma late mRNAs requires the presence of leader exons bordered by functional 3' and 5' splice sites, whether or not these sites are used during pre-mRNA processing.

Base Sequence↗
PubMed1992

Group I permuted intron-exon (PIE) sequences self-splice to produce circular exons.

Circularly permuted group I intron precursor RNAs, containing end-to-end fused exons which interrupt half-intron sequences, were generated and tested for self-splicing activity. An autocatalytic RNA can form when the primary order of essential intron sequence elements, splice sites, and exons are permuted in this manner. Covalent attachment of guanosine to the 5' half-intron product, and accurate exon ligation indicated that the mechanism and specificity of splicing were not altered. However, because the exons were fused and the order of the splice sites reversed, splicing released the fused-exon as a circle. With this arrangement of splice sites, circular exon production was a prediction of the group I splicing mechanism. Circular RNAs have properties that would make them attractive for certain studies of RNA structure and function. Reversal of splice site sequences in a context that allows splicing, such as those generated by circularly permuted group I introns, could be used to generate short defined sequences of circular RNA in vitro and perhaps in vivo.

Anabaena↗
PubMed1992

Mutations which alter splicing in the human hypoxanthine-guanine phosphoribosyltransferase gene.

A large proportion of mutations at the human hprt locus result in aberrant splicing of the hprt mRNA. We have been able to relate the mutation to the splicing abnormality in 30 of these mutants. Mutations at the splice acceptor sites of introns 4, 6 and 7 result in splicing out of the whole of the downstream exons, whereas in introns 1, 7 or 8 a cryptic site in the downstream exon can be used. Mutations in the donor site of introns 1 and 5 result in the utilisation of cryptic sites further downstream, whereas in the other introns, the upstream exons are spliced out. Our most unexpected findings were mutations in the middle of exons 3 and 8 which resulted in splicing out of these exons in part of the mRNA populations. Our results have enabled us to assess current models of mRNA splicing. They emphasize the importance of the polypyrimidine tract in splice acceptor sites, they support the role of the exon as the unit of assembly for splicing, and they are consistent with a model proposing a stem-loop structure for exon 8 in the hprt mRNA.

Base Sequence↗
PubMed1992

Phosphorothioate substitution identifies phosphate groups important for pre-mRNA splicing.

Substitution of pre-mRNA in vitro splicing substrates with alpha-phosphorothioate ribonucleotide analogs has multiple effects on the processes of spliceosome formation and splicing. A major effect of substitution is on the splicing cleavage/ligation reactions. Substitution at the 5' splice junction blocks the first cleavage/ligation reaction while substitution at the 3' splice junction blocks the second cleavage/ligation reaction. A second effect of phosphorothioate substitution is the inhibition of spliceosome formation. A substitution/interference assay was used to determine positions where substitution inhibits spliceosome formation or splicing. Substitution in the 3' splice site polypyrimidine tract was found to inhibit spliceosome formation and splicing. This effect was enhanced with multiple substitutions in the region. No sites of substitution within the exons were found which affected spliceosome formation or splicing.

Adenoviridae↗