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At least 325 records · Page 18Linked to original sources
PubMed1999

Binding of hnRNP H to an exonic splicing silencer is involved in the regulation of alternative splicing of the rat beta-tropomyosin gene.

In the rat beta-tropomyosin (beta-TM) gene, exons 6 and 7 are spliced alternatively in a mutually exclusive manner. Exon 6 is included in mRNA encoding nonmuscle TM-1, whereas exon 7 is used in mRNA encoding skeletal muscle beta-TM. Previously, we demonstrated that a six nucleotide mutation at the 5' end of exon 7, designated as ex-1, activated exon 7 splicing in nonmuscle cells. In this study, we show that the activating effect of this mutation is not the result of creating an exonic splicing enhancer (ESE) or disrupting a putative secondary structure. The sequence in exon 7 acts as a bona fide exonic splicing silencer (ESS), which is bound specifically by a trans-acting factor. Isolation and peptide sequencing reveal that this factor is hnRNP H, a member of the heterogeneous nuclear ribonucleoprotein (hnRNP) family. Binding of hnRNP H correlates with the ESS activity. Furthermore, addition of antibodies that specifically recognizes hnRNP H to the splicing reactions or partial depletion of hnRNP H from nuclear extract activates exon 7 splicing in vitro and this effect can be reversed by addition of purified recombinant hnRNP H. These results indicate that hnRNP H participates in exclusion of exon 7 in nonmuscle cells. The involvement of hnRNP H in the activity of an ESS may represent a prototype for the regulation of tissue- and developmental-specific alternative splicing.

Alternative Splicing↗
PubMed1990

Regulation of sex-specific RNA splicing at the Drosophila doublesex gene: cis-acting mutations in exon sequences alter sex-specific RNA splicing patterns.

Sex-specific alternative RNA splicing of the doublesex (dsx) pre-mRNA results in sex-specific polypeptides that regulate both male and female somatic sexual differentiation in Drosophila melanogaster. We have molecularly characterized a class of dsx mutations that act in cis to disrupt the regulation of dsx RNA processing, causing the dsx pre-mRNA to be spliced in the male-specific pattern regardless of the chromosomal sex of the fly. These dsx mutations are associated with rearrangements in the female-specific exon just 3' to the female-specific splice acceptor. The mutations do not affect the female-specific splice sites or intron that are identical to wild-type sequences. These results indicate that sequences in the female-specific exon are important for the regulation of sex-specific RNA splicing, perhaps by acting as sites of interaction with trans-acting regulators. Furthermore, the data suggest that female-specific regulation of dsx RNA processing occurs by promoting the usage of the female splice acceptor site, rather than by repressing the usage of the alternative male-specific splice acceptor.

Amino Acid Sequence↗
PubMed1996

The Moloney murine sarcoma virus ts110 5' splice site signal contributes to the regulation of splicing efficiency and thermosensitivity.

The 5' splice site signal (5'ss) in Moloney murine sarcoma virus ts110 (MuSVts110) RNA was found to participate in the regulation of its splicing phenotype. This 5'ss (CAG/GUAGGA) departs from the mammalian consensus (CAG/GURAGU) at positions +4 and +6, both of which base pair with U1 and U6 small nuclear RNAs during splicing. A doubling in splicing efficiency and near elimination of the splicing thermosensitivity characteristic of MuSVts110 were observed in 5'ss mutants containing a U at position +6 (termed 5' A6U), even in those in which U1-5'ss complementarity had been reduced. At the permissive temperature (28 degrees C), the 5' A6U mutation increased the efficiency of the second splicing reaction, while at the nonpermissive temperature (39 degrees C), both splicing reactions were positively affected.

3T3 Cells↗
PubMed1990

Myosin light-chain 1/3 gene alternative splicing: cis regulation is based upon a hierarchical compatibility between splice sites.

The mechanisms involved in the selective joining of appropriate 5' and 3' splice sites are still poorly understood in both constitutive and alternatively spliced genes. With two promoters associated with different exons, the myosin light-chain 1/3 gene generates two pre-mRNAs that also differ by the use of a pair of internal exons, 3 and 4, that are spliced in a mutually exclusive fashion. When the promoter upstream from exon 1 is used, only exon 4 is included. If the promoter upstream from exon 2 is used, only exon 3 is included. In an attempt to understand the molecular basis for the mutually exclusive behavior of these two exons and the basis of their specific selection, a number of minigene constructs containing exons 3 and 4 were tested in a variety of homologous or heterologous cis and trans environments. The results demonstrate that the mutually exclusive behavior of myosin light-chain exons 3 and 4 and selection between the two exons are cis regulated and are affected by the nature of the flanking sequences. Both exons competed for the common flanking 5' and 3' splice sites. Flanking exons were found that favored inclusion into mature mRNA of exon 3, exon 4, both, or neither, suggesting a specific cooperative interaction between certain 5' and 3' splice sites. Thus, alternative splicing of myosin light-chain 1/3 pre-mRNAs is regulated in cis by a hierarchy of compatibilities between pairs of 5' and 3' splice sites.

Animals↗
PubMed1990

Base pairing between the 3' exon and an internal guide sequence increases 3' splice site specificity in the Tetrahymena self-splicing rRNA intron.

It has been proposed that recognition of the 3' splice site in many group I introns involves base pairing between the start of the 3' exon and a region of the intron known as the internal guide sequence (R. W. Davies, R. B. Waring, J. Ray, T. A. Brown, and C. Scazzocchio, Nature [London] 300:719-724, 1982). We have examined this hypothesis, using the self-splicing rRNA intron from Tetrahymena thermophila. Mutations in the 3' exon that weaken this proposed pairing increased use of a downstream cryptic 3' splice site. Compensatory mutations in the guide sequence that restore this pairing resulted in even stronger selection of the normal 3' splice site. These changes in 3' splice site usage were more pronounced in the background of a mutation (414A) which resulted in an adenine instead of a guanine being the last base of the intron. These results show that the proposed pairing (P10) plays an important role in ensuring that cryptic 3' splice sites are selected against. Surprisingly, the 414A mutation alone did not result in activation of the cryptic 3' splice site.

Animals↗
PubMed2002

Regulation of corticotropin-releasing hormone type 2 receptors by multiple promoters and alternative splicing: identification of multiple splice variants.

We demonstrate that multiple promoters and alternate splicing regulate expression of the human CRH receptor type 2 (CRHR2) gene. We show that flanking regions to the first exons drive promoter activity in both endogenously and nonendogenously expressing cell lines. Putative promoter elements have been identified that are conserved between species, including the comparison of CRHR2gamma in nonhuman primates that was previously known only in humans, which may be responsible for subtype tissue specific regulation. We have identified novel transcripts produced by alternate splicing of the first exon of CRHR2beta (beta1a) with various combinations of the 5' exons including a novel exon (beta1c) spliced to the common exons. The 5' structure of the gene permits many other combinations of alternate splicing that may arise as part of a regulatory mechanism controlling functional receptor expression. The 5'-untranslated region of the first exons has been extended; and 3' acceptor sites identified within the 5' untranslated region of CRHR2gamma and CRHR2alpha are used during alternate splicing of CRHR2beta upstream exons. This has important implications because various reports on the expression of CRHR2gamma and CRHR2alpha have been unable to discriminate between the functional receptor and CRHR2beta alternate splice variants. Only the described sequences upstream of the 3' splice site are unique to CRHR2gamma and CRHR2alpha.

5' Untranslated Regions↗
PubMed2005

Analysis of a splice array experiment elucidates roles of chromatin elongation factor Spt4-5 in splicing.

Splicing is an important process for regulation of gene expression in eukaryotes, and it has important functional links to other steps of gene expression. Two examples of these linkages include Ceg1, a component of the mRNA capping enzyme, and the chromatin elongation factors Spt4-5, both of which have recently been shown to play a role in the normal splicing of several genes in the yeast Saccharomyces cerevisiae. Using a genomic approach to characterize the roles of Spt4-5 in splicing, we used splicing-sensitive DNA microarrays to identify specific sets of genes that are mis-spliced in ceg1, spt4, and spt5 mutants. In the context of a complex, nested, experimental design featuring 22 dye-swap array hybridizations, comprising both biological and technical replicates, we applied five appropriate statistical models for assessing differential expression between wild-type and the mutants. To refine selection of differential expression genes, we then used a robust model-synthesizing approach, Differential Expression via Distance Synthesis, to integrate all five models. The resultant list of differentially expressed genes was then further analyzed with regard to select attributes: we found that highly transcribed genes with long introns were most sensitive to spt mutations. QPCR confirmation of differential expression was established for the limited number of genes evaluated. In this paper, we showcase splicing array technology, as well as powerful, yet general, statistical methodology for assessing differential expression, in the context of a real, complex experimental design. Our results suggest that the Spt4-Spt5 complex may help coordinate splicing with transcription under conditions that present kinetic challenges to spliceosome assembly or function.

Chromatin↗
PubMed2000

Novel transcripts of fibroblast growth factor receptor 3 reveal aberrant splicing and activation of cryptic splice sequences in colorectal cancer.

A nested reverse transcription-PCR analysis of FGFR3 from human colorectal carcinomas revealed novel mutant transcripts caused by aberrant splicing and activation of cryptic splice sequences. Two aberrantly spliced transcripts were detected with high frequency in 50% of 36 primary tumors and in 60% of 10 human colorectal cancer cell lines. Most transcripts used normal splice sites but skipped or included exons 8 and 9. Two mutant transcripts arose from cryptic splice donor sites in exon 7 that spliced to exon 10. The predicted translation products would exhibit frameshifts and a premature termination codon in exon 10. We propose that dysregulation of mRNA splicing frequently generates an aberrant FGFR3 transcript that may confer a selectable advantage on clones of cells in colorectal tumorigenesis.

Alternative Splicing↗
PubMed1992

In vitro splicing of cardiac troponin T precursors. Exon mutations disrupt splicing of the upstream intron.

A single cardiac troponin T (cTNT) gene generates two mRNAs by including or excluding the 30-nucleotide exon 5 during pre-mRNA processing. Transfection analysis of cTNT minigenes has previously demonstrated that both mRNAs are expressed from unmodified minigenes, and mutations within exon 5 can lead to complete skipping of the exon. These results suggested a role for exon sequence in splice site recognition. To investigate this potential role, an in vitro splicing system using cTNT precursors has been established. Two-exon precursors containing the alternative exon and either the upstream exon or downstream exon were spliced accurately and efficiently in vitro. The mutations within the alternative exon that resulted in exon skipping in vivo specifically blocked splicing of the upstream intron in vitro and had no effect on removal of the downstream intron. In addition, the splicing intermediates of these two precursors have been characterized, and the branch sites utilized on the introns flanking the alternative exon have been determined. Potential roles of exon sequence in splice site selection are discussed. These results establish a system that will be useful for the biochemical characterization of the role of exon sequence in splice site selection.

Animals↗
PubMed1990

A difference in the splicing patterns of the closely related normal and variant human growth hormone gene transcripts is determined by a minimal sequence divergence between two potential splice-acceptor sites.

The human growth hormone (hGH-N) and the closely related human growth hormone-variant (hGH-V) genes differ in their patterns of splice-site selection. In 9% of hGH-N transcripts exon 2 is spliced to an alternative acceptor site located 45 bases within exon 3. mRNA spliced in this manner encodes a 20-kDa hGH-N isoform instead of the normal 22-kDa hGH-N. The hGH-V transcript fails to utilize this alternative splicing pathway. A region of the hGH-N and hGH-V genes critical to this difference in splice-site selection has been identified by reciprocal exchange of corresponding genomic fragments and has been defined in detail by a series of reciprocal site-specific exchanges. Three base differences located between the two potential splice-acceptor sites are both necessary and sufficient in defining the respective splicing patterns. One of these bases may serve as a lariat branch point critical for the alternative acceptor site activity while the remaining two bases appear to modulate the frequency with which this site is selected.

Animals↗
PubMed1995

Titration of serine/arginine (SR) splicing factors during adenoviral infection modulates E1A pre-mRNA alternative splicing.

Alternative splicing of the adenovirus-2 E1A pre-mRNA involves the use of three 5' splice sites and is modulated during infection because the 13S mRNA and 9S mRNA reactions are predominant during the early and late periods, respectively. We had previously reproduced in vitro the 13S to 9S modulation with nuclear extracts isolated from infected HeLa cells and shown that high molecular weight viral RNAs are involved in this modulation, most likely by sequestering or titrating general splicing factors. To further test this hypothesis, we titrated splicing factors from an uninfected nuclear extract using competitor RNA or by progressive inactivation of splicing factors with monoclonal antibodies. We found that the 13S to 9S modulation occurs when titrating only with certain RNAs (essentially adenoviral RNAs), and also by progressively inactivating the 9G8 SR splicing factor. The demonstration that late nuclear extracts contain levels of active SR splicing factors limiting for the 13S reaction has been made by complementation experiments. We show that late nuclear extracts do not complement SR factor-deficient extracts, whereas late extracts treated with micrococcal nuclease complement them. Furthermore, complementation of late nuclear extracts with each of the three 30-35-kDa SR factors (9G8, SC35, and SF2/ASF) restores an efficient 13S mRNA reaction. Thus, our results provide evidence that the 13S to 9S modulation is triggered through a titration of SR factors required for the 13S mRNA reaction by major late transcripts that accumulate in nuclei late in infection.

Adenoviridae↗
PubMed1996

Cis-acting elements distinct from the 5' splice site promote U1-independent pre-mRNA splicing.

We have identified a class of pre-mRNAs that are spliced in HeLa extracts depleted for U1 snRNP (delta U1 extracts). Previously, we described pre-mRNAs that can be spliced in delta U1 extracts only when high concentrations of SR splicing factors are added. In contrast, the substrates characterized here are efficiently processed in delta U1 extracts without the addition of excess SR proteins. The members of this class comprise both a naturally occurring pre-mRNA, from the Drosophila fushi tarazu gene, and a chimera containing sequences from two different pre-mRNAs that individually are dependent upon U1 snRNP or excess SR proteins. Several sequence elements account for the variations in dependence on U1 snRNP and SR proteins for splicing. In one pre-mRNA, a single element was identified adjacent to the branch site. In the other, two elements flanking the 5' splice site were found to be critical. This U1-independent splicing reaction may provide a mechanism for cells to control the extent of processing of different classes of pre-mRNAs in response to altered activities of SR proteins, and furthermore suggests that U1 snRNP-independent splicing may not be uncommon.

Base Sequence↗
PubMed1997

The SR splicing factors ASF/SF2 and SC35 have antagonistic effects on intronic enhancer-dependent splicing of the beta-tropomyosin alternative exon 6A.

Exons 6A and 6B of the chicken beta-tropomyosin gene are mutually exclusive and selected in a tissue-specific manner. Exon 6A is present in non-muscle and smooth muscle cells, while exon 6B is present in skeletal muscle cells. In this study we have investigated the mechanism underlying exon 6A recognition in non-muscle cells. Previous reports have identified a pyrimidine-rich intronic enhancer sequence (S4) downstream of exon 6A as essential for exon 6A 5'-splice site recognition. We show here that preincubation of HeLa cell extracts with an excess of RNA containing this sequence specifically inhibits exon 6A recognition by the splicing machinery. Splicing inhibition by an excess of this RNA can be rescued by addition of the SR protein ASF/SF2, but not by the SR proteins SC35 or 9G8. ASF/SF2 stimulates exon 6A splicing through specific interaction with the enhancer sequence. Surprisingly, SC35 behaves as an inhibitor of exon 6A splicing, since addition to HeLa nuclear extracts of increasing amounts of the SC35 protein completely abolish the stimulatory effect of ASF/SF2 on exon 6A splicing. We conclude that exon 6A recognition in vitro depends on the ratio of the ASF/SF2 to SC35 SR proteins. Taken together our results suggest that variations in the level or activity of these proteins could contribute to the tissue-specific choice of beta-tropomyosin exon 6A. In support of this we show that SR proteins isolated from skeletal muscle tissues are less efficient for exon 6A stimulation than SR proteins isolated from HeLa cells.

Alternative Splicing↗
PubMed1999

Recognition of exonic splicing enhancer sequences by the Drosophila splicing repressor RSF1.

The Drosophila repressor splicing factor 1 (RSF1) comprises an N-terminal RNA-binding region and a C-terminal domain rich in glycine, arginine and serine residues, termed the GRS domain. Recently, RSF1 has been shown to antagonize splicing factors of the serine/arginine-rich (SR) family and it is, therefore, expected to play a role in processing of a subset of Drosophila pre-mRNAs through specific interactions with RNA. To investigate the RNA-binding specificity of RSF1, we isolated RSF1-binding RNAs using an in vitro selection approach. We have identified two RNA target motifs recognized by RSF1, designated A (CAACGACGA)- and B (AAACGCGCG)-type sequences. We show here that the A-type cognate sequence behaves as an SR protein-dependent exonic splicing enhancer. Namely, three copies of the A-type ligand bind SR proteins, stimulate the efficiency of splicing of reporter pre-mRNAs several fold and lead to inclusion of a short internal exon both in vitro and in vivo. However, three copies of a B-type ligand were much less active. The finding that RSF1 acts as a potent repressor of pre-mRNA splicing in vitro led us to propose that the equilibrium between a limited number of structurally-related general splicing activators or repressors, competing for common or promiscuous binding sites, may be a major determinant of the underlying mechanisms controlling many alternative pre-mRNA process-ing events.

Amino Acid Sequence↗
PubMed1994

The concentration of B52, an essential splicing factor and regulator of splice site choice in vitro, is critical for Drosophila development.

B52 is a Drosophila melanogaster protein that plays a role in general and alternative splicing in vitro. It is homologous to the human splicing factor ASF/SF2 which is essential for an early step(s) in spliceosome assembly in vitro and also regulates 5' and 3' alternative splice site choice in a concentration-dependent manner. In vitro, B52 can function as both a general splicing factor and a regulator of 5' alternative splice site choice. Its activity in vivo, however, is largely uncharacterized. In this study, we have further characterized B52 in vivo. Using Western blot (immunoblot) analysis and whole-mount immunofluorescence, we demonstrate that B52 is widely expressed throughout development, although some developmental stages and tissues appear to have higher B52 levels than others do. In particular, B52 accumulates in ovaries, where it is packaged into the developing egg and is localized to nuclei by the late blastoderm stage of embryonic development. We also overexpressed this protein in transgenic flies in a variety of developmental and tissue-specific patterns to examine the effects of altering the concentration of this splicing factor in vivo. We show that, in many cell types, changing the concentration of B52 adversely affects the development of the organism. We discuss the significance of these observations with regard to previous in vitro results.

Animals↗
PubMed2025

Improving spliced alignment by modeling splice sites with deep learning.

MOTIVATION: Spliced alignment refers to the alignment of messenger RNA (mRNA) or protein sequences to eukaryotic genomes. It plays a critical role in gene annotation and the study of gene functions. Accurate spliced alignment demands sophisticated modeling of splice sites, but current aligners use simple models, which may affect their accuracy given dissimilar sequences. RESULTS: We implemented minisplice to learn splice signals with a one-dimensional convolutional neural network (1D-CNN) and trained a model with 7,026 parameters for vertebrate and insect genomes. It captures conserved splice signals across phyla and reveals GC-rich introns specific to mammals and birds. We used this model to estimate the empirical splicing probability for every GT and AG in genomes, and modified minimap2 and miniprot to leverage pre-computed splicing probability during alignment. Evaluation on human long-read RNA-seq data and cross-species protein datasets showed our method greatly improves the junction accuracy especially for noisy long RNA-seq reads and proteins of distant homology. AVAILABILITY AND IMPLEMENTATION: https://github.com/lh3/minisplice.

Journal Article↗
PubMed1999

Mutational-screening in the factor VIII gene resulting in the identification of three novel mutations, one of which is a donor splice mutation. Mutations in brief no. 245. Online.

Hemophilia A is an X-linked bleeding disease caused by mutations in the coagulation factor VIII gene. The identification and characterization of pathogenic mutations allows the recognition of new mechanisms of functional disturbances of factor VIII. To screen for mutations exons 1-26 of the factor VIII gene have been amplified genomically and analyzed by SSCP followed by direct sequencing of respective exons showing abnormal electrophoretic mobility on SSCP analysis. In the present study we report the detection of four mutations in the factor VIII gene, of which three are novel. The mutational analysis of a patient with severe hemophilia A has revealed that the ac transversion at position 3 of the donor-splice-site of intron 23 results in the skipping of exon 23. A novel nonsense mutation Q1778X in exon 16 of factor VIII gene has been identified in a second hemophilia A case. Furthermore two missense mutations have been ascertained: a novel, S183R, causing a mild phenotype of hemophilia A and R282H, previously described in association with severe hemophilia A.

Exons↗
PubMed1997

The splicing factor SRp20 modifies splicing of its own mRNA and ASF/SF2 antagonizes this regulation.

SRp20 is a member of the highly conserved SR family of splicing regulators. Using a variety of reporter gene constructs, we show that SRp20 regulates alternative splicing of its own mRNA. Overexpression of SRp20 results in a reduction in the level of exon 4-skipped SRp20 transcripts and activates the production of transcripts containing exon 4. These exon 4-included transcripts encode a truncated protein lacking the C-terminal RS domain. We provide evidence that SRp20 probably enhances the recognition of the otherwise unused, weak splice acceptor of exon 4. The recognition of exons with weak splice acceptor sites may be a general activity of SRp20. Unexpectedly, ASF/SF2, another member of the SR family, antagonizes the effect of SRp20 on SRp20 pre-mRNA splicing and suppresses the production of the exon 4-included form. Our results indicate that ASF/SF2 suppresses the use of the alternative exon 4, most likely by inhibiting the recognition of the splice donor of exon 4. These results demonstrate, for the first time, an auto-regulatory activity of an SR protein which is antagonized by a second SR protein.

Alternative Splicing↗