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PubMed1999

Splicing defects in the ataxia-telangiectasia gene, ATM: underlying mutations and consequences.

Mutations resulting in defective splicing constitute a significant proportion (30/62 [48%]) of a new series of mutations in the ATM gene in patients with ataxia-telangiectasia (AT) that were detected by the protein-truncation assay followed by sequence analysis of genomic DNA. Fewer than half of the splicing mutations involved the canonical AG splice-acceptor site or GT splice-donor site. A higher percentage of mutations occurred at less stringently conserved sites, including silent mutations of the last nucleotide of exons, mutations in nucleotides other than the conserved AG and GT in the consensus splice sites, and creation of splice-acceptor or splice-donor sites in either introns or exons. These splicing mutations led to a variety of consequences, including exon skipping and, to a lesser degree, intron retention, activation of cryptic splice sites, or creation of new splice sites. In addition, 5 of 12 nonsense mutations and 1 missense mutation were associated with deletion in the cDNA of the exons in which the mutations occurred. No ATM protein was detected by western blotting in any AT cell line in which splicing mutations were identified. Several cases of exon skipping in both normal controls and patients for whom no underlying defect could be found in genomic DNA were also observed, suggesting caution in the interpretation of exon deletions observed in ATM cDNA when there is no accompanying identification of genomic mutations.

Ataxia Telangiectasia↗

PubMed2002

Functional inactivation of the SR family of splicing factors during a vaccinia virus infection.

SR proteins are essential splicing factors required for constitutive splicing and function as key regulators of alternative RNA splicing. We have shown that SR proteins purified from late adenovirus-infected cells (SR-Ad) are functionally inactivated as splicing enhancer or splicing repressor proteins by a virus-induced partial de-phosphorylation. Here, we show that SR proteins purified from late vaccinia-virus-infected cells (SR-VV) are also hypo-phosphorylated and functionally inactivated as splicing regulatory proteins. We further show that incubating SR-Ad proteins under conditions that restore the phospho-epitopes to the SR proteins results in the restoration of their activity as splicing enhancer and splicing repressor proteins. Interestingly, re-phosphorylation of SR-VV proteins only partially restored the splicing enhancer or splicing repressor phenotype to the SR proteins. Collectively, our results suggest that viral control of SR protein activity may be a common strategy used by DNA viruses to take control of the host cell RNA splicing machinery.

Adenoviridae↗

PubMed1986

Splice site consensus sequences are preferentially accessible to nucleases in isolated adenovirus RNA.

The conformation of RNA sequences spanning five 3' splice sites and two 5' splice sites in adenovirus mRNA was probed by partial digestion with single-strand specific nucleases. Although cleavage of nucleotides near both 3' and 5' splice sites was observed, most striking was the preferential digestion of sequences near the 3' splice site. At each 3' splice site a region of very strong cleavage is observed at low concentrations of enzyme near the splice site consensus sequence or the upstream branch point consensus sequence. Additional sites of moderately strong cutting near the branch point consensus sequence were observed in those sequences where the splice site was the preferred target. Since recognition of the 3' splice site and branch site appear to be early events in mRNA splicing these observations may indicate that the local conformation of the splice site sequences may play a direct or indirect role in enhancing the accessibility of sequences important for splicing.

Adenovirus Early Proteins↗

PubMed1988

Cis requirements for alternative splicing of the cardiac troponin T pre-mRNA.

The cardiac troponin T (cTNT) pre-mRNA splices 17 exons contiguously but alternatively splices (includes or excludes) the fifth exon. Because both alternative splice products are processed from the same pre-mRNA species, the cTNT pre-mRNA must contain cis-acting sequences which specify exon 5 as an alternative exon. A cTNT minigene (SM-1) transfected into cultured cells produces mRNAs both including and excluding exon 5. The junctions of exons 4-5-6 and 4-6 in the cTNT minigene mRNAs are identical to those of endogenous cTNT mRNAs and no other exons are alternatively spliced. Thus, the SM-1 pre-mRNA is correctly alternatively spliced in transfected cells. To circumscribe the pre-mRNA regions which are required for the alternative nature of exon 5, we have constructed a systematic series of deletion mutants of SM-1. Transfection of this series demonstrates that a 1200 nt pre-mRNA region containing exons 4, 5, and 6 is sufficient to direct alternative splicing of exon 5. Within this region are two relatively large inverted repeats which potentially sequester the alternative exon via intramolecular base-pairing. Such sequestration of an alternative exon is consistent with models which propose pre-mRNA conformation as being determinative for alternative splicing of some pre-mRNAs. However, deletion mutants which remove the majority of each of the inverted repeats retain the ability to alternatively splice exon 5 demonstrating that neither is required for cTNT alternative splice site selection. Taken together, deletion analysis has limited cis elements required for alternative splicing to three small regions of the pre-mRNA containing exons 4, 5, and 6. In addition, the cTNT minigene pre-mRNA expresses both alternative splice products in a wide variety of cultured non-muscle cells as well as in cultured striated muscle cells, although expression of the cTNT pre-mRNA is normally restricted to striated muscle. This indicates that cis elements involved in defining the cTNT exon 5 as an alternative exon do not require muscle-specific factors in trans to function.

Animals↗

PubMed1990

The molecular basis for alternative splicing of the CABP1 transcripts in Dictyostelium discoideum.

We have determined the nucleotide sequence of the CABP1 gene from Dictyostelium discoideum. Together with previous data on cDNA sequences, we establish that alternative splicing of transcripts derived from this gene is responsible for the production of the two CABP1 subunits. RNA blot analysis suggested that alternative splicing of the CABP1 transcripts occurs during growth and throughout development. In addition, we have compiled the intron sequences of Dictyostelium pre-mRNAs and observed that the GUAAGU hexanucleotide at the 5' splice site is highly conserved. The 5' splice site of CABP1 deviates from the consensus hexanucleotide in having a sequence of GUAAUA. To assess the role of the modified 5' splice on differential splicing, we have constructed an actin-CABP1 fusion gene and transformed it into Dictyostelium cells. Analysis by immunoprecipitation, with anti-CABP1 antibody and amplification of specific cDNAs by polymerase chain reaction show that the transcripts generated by the fusion gene are alternatively spliced. When the 5' splice site of the fusion gene is mutated to conform to the consensus sequence, the resulting transcripts are constitutively spliced. These observations suggest that changes in positions 5 and 6 of the donor splice site are involved in the alternative splicing of the CABP1 transcripts.

Actins↗

PubMed1993

Functional analysis of a C. elegans trans-splice acceptor.

The rol-6 gene is trans-spliced to the 22 nt leader, SL1, 173 nt downstream of the transcription start. We have analyzed splicing in transformants carrying extrachromosomal arrays of rol-6 with mutations in the trans-splice acceptor site. This site is a close match to the consensus, UUUCAG, that is highly conserved in both trans-splice and intron acceptor sites in C. elegans. When the trans-splice site was inactivated by mutating the perfectly-conserved AG, trans-splicing still occurred, but at a cryptic site 20 nt upstream. We tested the frequency with which splicing switched from the normal site to the cryptic site when the pyrimidines at this site were changed to A's. Since most C. elegans 3' splice sites lack an obvious polypyrimidine tract, we hypothesized that these four pyrimidines might play this role, and indeed mutation of these bases caused splicing to switch to the cryptic site. We also demonstrated that a major reason the downstream site is normally favored is because it occurs at a boundary between A+U rich and non-A+U rich RNA. When the RNA between the two splice sites was made less A+U rich, splicing occurred preferentially at the upstream site.

Amino Acid Sequence↗

PubMed1996

Inverse splicing of a discontinuous pre-mRNA intron generates a circular exon in a HeLa cell nuclear extract.

We have recently reported the first example of inverse splicing of a eukaryotic pre-mRNA intron using a whole cell extract from the yeast Saccharomyces cerevisiae. The concomitant circularization of the exon in the course of this splicing reaction gave rise to the hypothesis that the circular RNA species, which had been recently discovered in vivo in mammalian cells, were generated by inverse splicing. Here we report the formation of a circular exon in HeLa cell nuclear extracts by an inverse splicing reaction of the second intron of the human beta-globin gene from a pre-mRNA transcript in which the two intron halves flanked an artificially fused, single exon. Our data demonstrate that the mammalian pre-mRNA splicing system has indeed an intrinsic capability of aligning splice sites in reverse order and that this alignment can be followed by a complete splicing reaction, whereby the discontinuous intron sequences are removed. Thus we propose that circular exons in vivo arise as a result of an inverse splicing reaction following the pairing of a 5' splice site with an upstream 3' splice site and that the frequency of this event is influenced by the presence and strength of other, competing splice sites.

Cell Extracts↗

PubMed2001

Multiple features contribute to efficient constitutive splicing of an unusually large exon.

Vertebrate internal exons are usually between 50 and 400 nt long; exons outside this size range may require additional exonic and/or intronic sequences to be spliced into the mature mRNA. The mouse polymeric immunoglobulin receptor gene has a 654 nt exon that is efficiently spliced into the mRNA. We have examined this exon to identify features that contribute to its efficient splicing despite its large size; a large constitutive exon has not been studied previously. We found that a strong 5' splice site is necessary for this exon to be spliced intact, but the splice sites alone were not sufficient to efficiently splice a large exon. At least two exonic sequences and one evolutionarily conserved intronic sequence also contribute to recognition of this exon. However, these elements have redundant activities as they could only be detected in conjunction with other mutations that reduced splicing efficiency. Several mutations activated cryptic 5' splice sites that created smaller exons. Thus, the balance between use of these potential sites and the authentic 5' splice site must be modulated by sequences that repress or enhance use of these sites, respectively. Also, sequences that enhance cryptic splice site use must be absent from this large exon.

Alternative Splicing↗

PubMed2002

Genome-wide detection of tissue-specific alternative splicing in the human transcriptome.

We have developed an automated method for discovering tissue-specific regulation of alternative splicing through a genome-wide analysis of expressed sequence tags (ESTs). Using this approach, we have identified 667 tissue-specific alternative splice forms of human genes. We validated our muscle-specific and brain-specific splice forms for known genes. A high fraction (8/10) were reported to have a matching tissue specificity by independent studies in the published literature. The number of tissue-specific alternative splice forms is highest in brain, while eye-retina, muscle, skin, testis and lymph have the greatest enrichment of tissue-specific splicing. Overall, 10-30% of human alternatively spliced genes in our data show evidence of tissue-specific splice forms. Seventy-eight percent of our tissue-specific alternative splices appear to be novel discoveries. We present bioinformatics analysis of several tissue-specific splice forms, including automated protein isoform sequence and domain prediction, showing how our data can provide valuable insights into gene function in different tissues. For example, we have discovered a novel kidney-specific alternative splice form of the WNK1 gene, which appears to specifically disrupt its N-terminal kinase domain and may play a role in PHAII hypertension. Our database greatly expands knowledge of tissue-specific alternative splicing and provides a comprehensive dataset for investigating its functional roles and regulation in different human tissues.

Alternative Splicing↗

PubMed2004

Mer1p is a modular splicing factor whose function depends on the conserved U2 snRNP protein Snu17p.

Mer1p activates the splicing of at least three pre-mRNAs (AMA1, MER2, MER3) during meiosis in the yeast Saccharomyces cerevisiae. We demonstrate that enhancer recognition by Mer1p is separable from Mer1p splicing activation. The C-terminal KH-type RNA-binding domain of Mer1p recognizes introns that contain the Mer1p splicing enhancer, while the N-terminal domain interacts with the spliceosome and activates splicing. Prior studies have implicated the U1 snRNP and recognition of the 5' splice site as key elements in Mer1p-activated splicing. We provide new evidence that Mer1p may also function at later steps of spliceosome assembly. First, Mer1p can activate splicing of introns that have mutated branch point sequences. Secondly, Mer1p fails to activate splicing in the absence of the non-essential U2 snRNP protein Snu17p. Thirdly, Mer1p interacts with the branch point binding proteins Mud2p and Bbp1p and the U2 snRNP protein Prp11p by two-hybrid assays. We conclude that Mer1p is a modular splicing regulator that can activate splicing at several early steps of spliceosome assembly and depends on the activities of both U1 and U2 snRNP proteins to activate splicing.

Base Sequence↗

PubMed2005

Evolutionarily conserved and diverged alternative splicing events show different expression and functional profiles.

To better decipher the functional impact of alternative splicing, we classified alternative splicing events in 10,818 pairs of human and mouse genes based on conservation at genome and transcript levels. Expression levels of conserved alternative splices in human and mouse expressed sequence tag databases show strong correlation, indicating that alternative splicing is similarly regulated in both species. A total of 43% (8921) of mouse alternative splices could be found in the human genome but not in human transcripts. Five of eleven tested mouse predictions were observed in human tissues, demonstrating that mouse transcripts provide a valuable resource for identifying alternative splicing events in human genes. Combining gene-specific measures of conserved and diverged alternative splicing with both gene classification based on Gene Ontology (GO) and microarray-determined gene expression in 52 diverse human tissues and cell lines, we found conserved alternative splicing most enriched in brain-expressed signaling pathways. Diverged alternative splicing is more prevalent in testis and cancerous cell line up-regulated processes, including protein biosynthesis, responses to stress and responses to endogenous stimuli. Using conservation as a surrogate for functional significance, these results suggest that alternative splicing plays an important role in enhancing the functional capacity of central nervous systems, while non-functional splicing more frequently occurs in testis and cell lines, possibly as a result of cellular stress and rapid proliferation.

Alternative Splicing↗

PubMed2000

Testing the "proto-splice sites" model of intron origin: evidence from analysis of intron phase correlations.

A few nucleotide sites of nuclear exons that flank introns are often conserved. A hypothesis has suggested that these sites, called "proto-splice sites," are remnants of recognition signals for the insertion of introns in the early evolution of eukaryotic genes. This notion of proto-splice sites has been an important basis for the insertional theory of introns. This hypothesis predicts that the distribution of proto-splice sites would determine the distribution of intron phases, because the positions of introns are just a subset of the proto-splice sites. We previously tested this prediction by examining the proportions of the phases of proto-splice sites, revealing nothing in these proportion distributions similar to observed proportions of intron phases. Here, we provide a second independent test of the proto-splice site hypothesis, with regard to its prediction that the proto-splice sites would mimic intron phase correlations, using a CDS database we created from GenBank. We tested four hypothetical proto-splice sites G / G, AG / G, AG / GT, and C/AAG / R. Interestingly, while G / G and AG / GT site phase distributions are not consistent with actual introns, we observed that AG / G and C/AAG / R sites have a symmetric phase excess. However, the patterns of the excess are quite different from the actual intron phase distribution. In addition, particular amino acid repeats in proteins were found to partially contribute to the excess of symmetry at these two types of sites. The phase associations of all four sites are significantly different from those of intron phases. Furthermore, a general model of intron insertion into proto-splice sites was simulated by Monte Carlo simulation to investigate the probability that the random insertion of introns into AG / G and C/AAG / R sites could generate the observed intron phase distribution. The simulation showed that (1) no observed correlation of intron phases was statistically consistent with the phase distribution of proto-splice sites in the simulated virtual genes; (2) most conservatively, no simulation in 10,000 Monte Carlo experiments gave a pattern with an excess of symmetric (1, 1) exons larger than those of (0, 0) and (2, 2), a major statistical feature of intron phase distribution that is consistent with the directly observed cases of exon shuffling. Thus, these results reject the null hypothesis that introns are randomly inserted into preexisting proto-splice sites, as suggested by the insertional theory of introns.

Animals↗

PubMed1987

A mutational analysis of spliceosome assembly: evidence for splice site collaboration during spliceosome formation.

We have analyzed the pathway of mammalian spliceosome assembly in vitro using a mobility retardation assay. The binding of splicing complexes to both wild-type and mutant beta-globin pre-RNAs was studied. Three kinetically related, ATP-dependent complexes, alpha, beta, and gamma, were resolved with a wild-type beta-globin substrate. These complexes formed, both temporally and in order of decreasing mobility, alpha----beta----gamma. All three complexes contained U2 snRNA. The RNA intermediates of splicing, i.e., free 5' exon and intron lariat + 3' exon, were found predominantly in the gamma complex. The RNA products of splicing, i.e., ligated exons and fully excised intron lariat, were found in separate, postsplicing complexes which appeared to form via breakdown of gamma. Mutations of the 5' splice site, which caused an accumulation of splicing intermediates, also resulted in accumulation of the gamma complex. Mutations of the 3' splice site, which severely inhibited splicing, reduced the efficiency and altered the pattern of complex formation. Surprisingly, the analysis of double mutants, with sequence alterations at both the 5' and 3' splice sites, revealed that the 5' splice site genotype was important for the efficient formation of a U2 snRNA-containing alpha complex at the 3' splice site. Thus, it appears that a collaborative interaction between the separate 5' and 3' splice sites promotes spliceosome assembly.

Animals↗

PubMed1990

Identification of two distinct intron elements involved in alternative splicing of beta-tropomyosin pre-mRNA.

The rat beta-tropomyosin gene encodes two isoforms, termed skeletal muscle beta-tropomyosin and fibroblast last tropomyosim 1 (TM-1), via an alternative RNA processing mechanism. The gene contains 11 exons. Exons 1-5 and exons 8 and 9 are common to all mRNAs expressed from the gene. Exons 6 and 11 are used in fibroblasts, as well as smooth muscle, whereas exons 7 and 10 are used only in skeletal muscle. In the present studies we focused on the mutually exclusive internal alternative splice choice involving exon 6 (fibroblast-type splice) and exon 7 (skeletal muscle-type splice). We have identified two distinct elements in the intron, upstream of exon 7, involved in splice site selection. The first element is comprised of a polypyrimidine tract located 89-143 nucleotides upstream of the 3' splice site, which specifies the location of the lariat branchpoints used, 144-153 nucleotides upstream of exon 7. The 3' splice site AG dinucleotide has no role in selection of these branchpoints. The second element is comprised of intron sequences located between the polypyrimidine tract and the 3' splice site of exon 7. It contains an important determinant in alternative splice site selection, because deletion of these sequences results in the use of the skeletal muscle-specific exon in nonmuscle cells. We propose that the use of lariat branchpoints located far upstream from a 3' splice site may be a general feature of some alternatively excised introns, reflecting the presence of regulatory sequences located between the lariat branch site and the 3' splice site. The data also indicate that alternative splicing of the rat beta-tropomyosin gene is regulated by a somewhat different mechanism from that described for rat alpha-tropomyosin gene and the transformer-2 gene of Drosophila melanogaster.

Animals↗

PubMed2005

Evolution of alternative splicing after gene duplication.

Alternative splicing and gene duplication are two major sources of proteomic function diversity. Here, we study the evolutionary trend of alternative splicing after gene duplication by analyzing the alternative splicing differences between duplicate genes. We observed that duplicate genes have fewer alternative splice (AS) forms than single-copy genes, and that a negative correlation exists between the mean number of AS forms and the gene family size. Interestingly, we found that the loss of alternative splicing in duplicate genes may occur shortly after the gene duplication. These results support the subfunctionization model of alternative splicing in the early stage after gene duplication. Further analysis of the alternative splicing distribution in human duplicate pairs showed the asymmetric evolution of alternative splicing after gene duplications; i.e., the AS forms between duplicates may differ dramatically. We therefore conclude that alternative splicing and gene duplication may not evolve independently. In the early stage after gene duplication, young duplicates may take over a certain amount of protein function diversity that previously was carried out by the alternative splicing mechanism. In the late stage, the gain and loss of alternative splicing seem to be independent between duplicates.

Alternative Splicing↗

PubMed2005

The serine/arginine-rich protein family in rice plays important roles in constitutive and alternative splicing of pre-mRNA.

Ser/Arg-rich (SR) proteins play important roles in the constitutive and alternative splicing of pre-mRNA. We isolated 20 rice (Oryza sativa) genes encoding SR proteins, of which six contain plant-specific characteristics. To determine whether SR proteins modulate splicing efficiency and alternative splicing of pre-mRNA in rice, we used transient assays in rice protoplasts by cotransformation of SR protein genes with the rice Waxy(b) (Wx(b))-beta-glucuronidase fusion gene. The results showed that plant-specific RSp29 and RSZp23, an SR protein homologous to human 9G8, enhanced splicing and altered the alternative 5' splice sites of Wx(b) intron 1. The resulting splicing pattern was unique to each SR protein; RSp29 stimulated splicing at the distal site, and RSZp23 enhanced splicing at the proximal site. Results of domain-swapping experiments between plant-specific RSp29 and SCL26, which is a homolog of human SC35, showed the importance of RNA recognition motif 1 and the Arg/Ser-rich (RS) domain for the enhancement of splicing efficiencies. Overexpression of plant-specific RSZ36 and SRp33b, a homolog of human ASF/SF2, in transgenic rice changed the alternative splicing patterns of their own pre-mRNAs and those of other SR proteins. These results show that SR proteins play important roles in constitutive and alternative splicing of rice pre-mRNA.

Alternative Splicing↗

PubMed1992

Intron excision from tRNA precursors by plant splicing endonuclease requires unique features of the mature tRNA domain.

It has been proposed that yeast and Xenopus splicing endonucleases initially recognize features in the mature tRNA domain common to all tRNA species and that the sequence and structure of the intron are only minor determinants of splice-site selection. In accordance with this postulation, we show that yeast endonuclease splices heterologous pre-tRNA(Tyr) species from vertebrates and plants which differ in their mature domains and intron secondary structures. In contrast, wheat germ splicing endonuclease displays a pronounced preference for homologous pre-tRNA species; an extensive study of heterologous substrates revealed that neither yeast pre-tRNA species specific for leucine, serine, phenylalanine and tyrosine nor human and Xenopus pre-tRNA(Tyr) species were spliced. In order to identify the elements essential for pre-tRNA splicing in plants, we constructed chimeric genes coding for tRNA precursors with a plant intron secondary structure and with mature tRNA(Tyr) domains from yeast and Xenopus, respectively. The chimeric pre-tRNA comprising the mature tRNA(Tyr) domain from Xenopus was spliced efficiently in wheat germ extract, whereas the chimeric construct containing the mature tRNA(Tyr) domain from yeast was not spliced at all. These data indicate that intron secondary structure contributes to the specificity of plant splicing endonuclease and that unique features of the mature tRNA domain play a dominant role in enzyme-substrate recognition. We further investigated the influence of specific nucleotides in the mature domain on splicing by generating a number of mutated pre-tRNA species. Our results suggest that nucleotides located in the D stem, i.e. in the center of the pre-tRNA molecule, are recognition points for plant splicing endonuclease.

Arabidopsis↗

PubMed1996

Alternative splicing of the pyruvate kinase M gene in a minigene system.

The M1-type and M2-type isozymes of pyruvate kinase are produced from a single gene by mutually exclusive use of exons 9 and 10. Selection of exon 10 generates the M2 type, which occurs in most tissues, whereas the M1 type is expressed by use of exon 9 only in skeletal muscle, heart and brain. We investigated the mechanism by which exon 10, but not exon 9 is selected in M2-expressing cells by transfecting minigenes containing exon 9 and/or exon 10 in cells and by analyzing the transcripts using reverse-transcriptase polymerase chain reaction. Deletion of the most conserved region in intron 8 did not affect selection of exon 10 in dRLh-84 cells, which express only the M2 type. Exclusion of exon 10 from the minigene resulted in two major spliced products. One included correctly spliced exon 9 and the other skipped this exon. Similar splicing patterns were observed when these minigenes were transfected in hepatocytes which express the L type, but not M1 or M2 types. The 5' splice site but not the 3' splice site of exon 9 was found to be hardly recognized by the splicing machinery in dRLh-84 cells. Mutation of the 5' splice site sequence of exon 9 to that of exon 10 and vice versa did not change the splicing patterns. However, mutation of this site of exon 9 to a perfectly complementary sequence of U1 snRNA resulted in selection of exon 9 correctly spliced to exon 10. A 9-10 fusion exon (constructed by substitution of 68 bases of the 3' portion of exon 9 and 33 bases of the 5' portion of intron 9 for the corresponding regions of exon 10 and intron 10) was also correctly incorporated into a major product together with exon 10. Thus, we propose that exon 9 is not recognized in non M1-expressing cells due to the weak signal of its 5' splice site and that, although the 5' splicing signal of exon 10 also appears to be weak, this exon can be recognized in these cells because the 5' recognition signal may be relatively strengthened by cis-acting element(s) which may be present in the 3' portion of exon 9 and the 5' portion of intron 9 and/or the corresponding regions of exon 10 and intron 10.

Alternative Splicing↗