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At least 469 records · Page 26Linked to original sources
PubMed2005

Molecular and genetic studies imply Akt-mediated signaling promotes protein kinase CbetaII alternative splicing via phosphorylation of serine/arginine-rich splicing factor SRp40.

Insulin regulates alternative splicing of PKCbetaII mRNA by phosphorylation of SRp40 via a phosphatidylinositol 3-kinase pathway (Patel, N. A., Chalfant, C. E., Watson, J. E., Wyatt, J. R., Dean, N. M., Eichler, D. C., and Cooper, D. C. (2001) J. Biol. Chem. 276, 22648-22654). Transient transfection of constitutively active Akt2 kinase promotes PKCbetaII exon inclusion. Serine/arginine-rich (SR) RNA-binding proteins regulating the selection of alternatively spliced exons are potential substrates of Akt kinase because many of them contain RXRXX(S/T) motifs. Here we show that Akt2 kinase phosphorylated SRp40 in vivo and in vitro. Mutation of Ser86 on SRp40 blocked in vitro phosphorylation. In control Akt2(+/+) fibroblasts, insulin treatment increased the phosphorylation of endogenous SR proteins, but their phosphorylation state remained unaltered by insulin in fibroblasts from Akt2(-/-) mice. Levels of PKCbetaII protein were up-regulated by insulin in Akt2(+/+) cells; however, only very low levels of PKCbetaII were detected in Akt2(-/-) cells and did not change following insulin treatment. Endogenous PKCbetaI and -betaII mRNA levels in Akt2(+/+) and Akt2(-/-) gastrocnemius muscle tissues were compared using quantitative real time PCR. The results indicated a 54% decrease in the expression of PKCbetaII levels in Akt(-/-), whereas PKCbetaI levels remained unchanged in both samples. Further, transfection of Akt2(-/-) cells with a PKCbetaII splicing minigene revealed defective betaII exon inclusion. Co-transfection of the mutated SRp40 attenuated betaII exon inclusion. This study provides in vitro and in vivo evidence showing Akt2 kinase directly phosphorylated SRp40, thereby connecting the insulin, PI 3-kinase/Akt pathway with phosphorylation of a site on a nuclear splicing protein promoting exon inclusion. This model is upheld in Akt2-deficient mice with insulin resistance leading to diabetes mellitus.

Alternative Splicing↗
PubMed1996

Base pairing at the 5' splice site with U1 small nuclear RNA promotes splicing of the upstream intron but may be dispensable for slicing of the downstream intron.

We previously reported that exon skipping in vivo due to point mutations in the 5' splice site (5'ss) signal of an internal mammalian exon can be prevented by coexpression of U1 small nuclear RNAs, termed shift-U1s, with complementarity to sequence upstream or downstream of the mutated site. We now show by S1 nuclease protection experiments that a typical shift-U1 restores splicing of the upstream intron, but not necessarily of the down stream intron. This indicates that the normal 5'ss sequence acts as an enhancer for splicing of the upstream intron, that it owes this activity to base pairing with U1, and that the enhancer activity is reproduced by base pairing of U1 with other sequences in the area. Shift-U1s are dispensable when the 3'ss sequence of the upstream intron is improved, which suggests that base pairing of U1 with sequences at or near the downstream end of the exon normally functions by compensating for a weakness in the upstream 3'ss. Accordingly, U1 appears to be involved in communication across the exon, but our data indicate at the same time that extensive base pairing between U1 and the 5'ss sequence is not necessary for accurate splicing of the downstream intron. These findings are discussed in relation to the coordinate selection exon termini proposed by the exon definition model.

Alternative Splicing↗
PubMed2006

Splicing factor Tra2-beta1 is specifically induced in breast cancer and regulates alternative splicing of the CD44 gene.

The human CD44 gene undergoes extensive alternative splicing of multiple variable exons positioned in a cassette in the middle of the gene. Expression of alternative exons is often restricted to certain tissues and could be associated with tumor progression and metastasis of several human malignancies, including breast cancer. Exon v4 contains multiple copies of a C/A-rich exon enhancer sequence required for optimal inclusion of the exon and binding to the nucleic acid-binding proteins YB-1 and human Tra2-beta1. Here, we show that hTra2-beta1, a member of the extended family of serine/arginine-rich (SR) splicing factors, enhances the in vivo inclusion of CD44 exons v4 and v5. It increased inclusion of exons v4 and v5 and acted synergistically with YB-1. Activation required the C/A-rich enhancer within exon v4. Several other SR proteins had none or only a slight effect on CD44 exon inclusion. In contrast, SC35 inhibited exon usage and antagonized the effects of Tra2 or YB-1. In a matched pair analysis of human breast cancers and their corresponding nonpathologic tissue controls, we found a significant induction of Tra2-beta1 in invasive breast cancer, both on the RNA and protein levels. Together with our functional data, these results suggest an important role for Tra2-beta1 in breast cancer. Induction of this splicing factor might be responsible for splicing of CD44 isoforms associated with tumor progression and metastasis.

Alternative Splicing↗
PubMed2002

Splicing regulation at the second catalytic step by Sex-lethal involves 3' splice site recognition by SPF45.

The Drosophila protein Sex-lethal (SXL) promotes skipping of exon 3 from its own pre-mRNA. An unusual sequence arrangement of two AG dinucleotides and an intervening polypyrimidine (Py)-tract at the 3' end of intron 2 is important for Sxl autoregulation. Here we show that U2AF interacts with the Py-tract and downstream AG, whereas the spliceosomal protein SPF45 interacts with the upstream AG and activates it for the second catalytic step of the splicing reaction. SPF45 represents a new class of second step factors, and its interaction with SXL blocks splicing at the second step. These results are in contrast with other known mechanisms of splicing regulation, which target early events of spliceosome assembly. A similar role for SPF45 is demonstrated in the activation of a cryptic 3' ss generated by a mutation that causes human beta-thalassemia.

Adenine Nucleotides↗
PubMed1994

Insertion of a T next to the donor splice site of intron 1 causes aberrantly spliced mRNA in a case of infantile GM1-gangliosidosis.

The lysosomal storage disorders GM1-gangliosidosis and Morquio B syndrome are caused by a complete or partial deficiency of acid beta-galactosidase. Here, we have characterized the mutation segregating in a family with two siblings affected by the severe infantile form of GM1-gangliosidosis. In total mRNA preparations derived from the patients' fibroblasts at least two aberrantly spliced beta-galactosidase transcripts (1 and 2) have been identified. Both transcripts contain a 20 nucleotide (nt) insertion derived from the 5' end of intron 1 of the beta-galactosidase gene. Furthermore, in transcript 2 sequences encoded by exon II are deleted during the splicing process. Comparison of the 20-nt insertion with wild-type intronic sequences indicated that in the genomic DNA of the patients an extra T nucleotide is present immediately downstream of the conserved GT splice donor dinucleotide of intron 1. Both patients are homozygous for the T nucleotide insertion. We propose that this single base insertion is the mutation responsible for aberrant splicing of beta-galactosidase pre-mRNA, giving rise to transcripts that cannot encode a normal protein.

Base Sequence↗
PubMed2000

Gene structure prediction by spliced alignment of genomic DNA with protein sequences: increased accuracy by differential splice site scoring.

Gene identification in genomic DNA from eukaryotes is complicated by the vast combinatorial possibilities of potential exon assemblies. If the gene encodes a protein that is closely related to known proteins, gene identification is aided by matching similarity of potential translation products to those target proteins. The genomic DNA and protein sequences can be aligned directly by scoring the implied residues of in-frame nucleotide triplets against the protein residues in conventional ways, while allowing for long gaps in the alignment corresponding to introns in the genomic DNA. We describe a novel method for such spliced alignment. The method derives an optimal alignment based on scoring for both sequence similarity of the predicted gene product to the protein sequence and intrinsic splice site strength of the predicted introns. Application of the method to a representative set of 50 known genes from Arabidopsis thaliana showed significant improvement in prediction accuracy compared to previous spliced alignment methods. The method is also more accurate than ab initio gene prediction methods, provided sufficiently close target proteins are available. In view of the fast growth of public sequence repositories, we argue that close targets will be available for the majority of novel genes, making spliced alignment an excellent practical tool for high-throughput automated genome annotation.

Algorithms↗
PubMed1998

Alternative splicing of human NrCAM in neural and nonneural tissues.

Neural cell adhesion molecule NrCAM exists in a variety of isoforms as a result of alternative splicing of individual exons during RNA processing. In this report we demonstrate that many of the alternative splicing events described for chick are conserved in man and describe a novel variant of NrCAM cDNA. Furthermore, we show that NrCAM is expressed at significant levels outside the nervous system; in particular in pancreas, adrenal glands, and placenta and that expression in both brain and other tissues is accompanied by a very variable pattern of exon utilization in fetal and adult cells.

Adrenal Glands↗
PubMed2002

Rous sarcoma virus negative regulator of splicing selectively suppresses SRC mRNA splicing and promotes polyadenylation.

Retroviruses require a balance of spliced and unspliced RNA for efficient replication. Here, we examined the effect of mutations in a splicing suppressor sequence called the negative regulator of splicing (NRS), located within the gag gene of Rous sarcoma virus. While the NRS mutant viruses showed only small changes in the levels of spliced env mRNAs, they had significant increases in src mRNA levels and transformed cells more efficiently than wild-type virus. None of these mutations prevented viral replication; however, some of the mutant viruses replicated more slowly than wild-type virus. In addition, increased transcriptional readthrough of the poly(A) site in the 3' LTR was observed with the NRS mutant viruses, suggesting that the wild-type NRS sequence promotes polyadenylation.

Animals↗
PubMed1989

A novel H-2K splice form: predictions for other alternative H-2 splicing events.

A large number of H-2K and H-2D cDNA clones from a C3HfB/HeN spleen cDNA library were extensively characterized. All H-2Dk cDNAs were shown to exhibit the short form of exon 8, consistent with the presence of a single lariat branchpoint site within intron 7. Twenty-five H-2Kkm2 cDNAs were found to bear a short exon 8, whereas only two clones were shown to carry the longer form of this exon. In one of the H-2Kkm2 cDNAs, a novel pattern of H-2 splicing was identified, in which an extra 15 nucleotides, derived from the 3' end of intron 5, were inserted between the intact and unaltered exon 5 and exon 6 sequences. Resulting from the apparent use of a cryptic splice acceptor site in place of the canonical intron 5 site, this insertion is predicted to generate an in-frame insertion of five nonpolar amino acid residues within a highly polar region of the intracytoplasmic domain of the H-2K polypeptide. The features of this novel splice form served as the basis for predicting additional rare, alternative H-2 pre-mRNA splicing events that might produce functionally relevant microheterogeneity in the encoded H-2 gene products.

Animals↗
PubMed1999

Analysis of splice variants of the fibronectin gene in thyroid carcinomas by reverse transcription-polymerase chain reaction: increased expression of oncofetal fibronectin mRNA in papillary carcinomas is not caused by the alternation in splicing.

The expression levels of each splice variant of the fibronectin gene in the normal thyroid and in thyroid tumors were examined by reverse transcription-polymerase chain reaction (RT-PCR). In thyroid papillary carcinomas, insertion of a variant exon in the ED-A and ED-B domains, and three of five types of splice variants in the IIICS domain were observed. In spite of the marked increase in the expression of oncofetal fibronectin mRNA with the IIICS sequence in papillary and anaplastic carcinomas in the previous reports, the relative expression levels of each splice variant with or without the IIICS sequence showed no difference among all the tumor types. Therefore, the much increased expression of oncofetal fibronectin mRNA in these carcinomas is not caused by the alternation in splicing, but may be caused by an increase in promoter activity or stability of mRNA of the fibronectin gene.

Adenocarcinoma, Follicular↗
PubMed2000

Illegitimate splicing of the NF1 gene in healthy individuals mimics mutation-induced splicing alterations in NF1 patients.

Neurofibromatosis type 1 (NF1) is a common inherited disease affecting one in 3,500 individuals. The mutation rate in the NF1 gene is one of the highest known for human genes. Compared to other methods, the protein truncation test (PTT) and subsequent sequence analysis of cloned cDNA provides improved efficiency in detecting NF1 mutations that are dispersed throughout the gene spanning 350 kb of genomic DNA. Sequencing of cDNA of patients affected with NF1 mutations revealed multiple splicing errors. Since similar missplicings were also found in "aged" blood of healthy individuals, they are most likely attributable to a general decrease in splice site selection in aged blood. We show that restoring viability of lymphocytes before RNA extraction by cultivation and PHA stimulation diminishes aberrant splicing in aged blood and is thus useful to circumvent splicing alterations which are frequently compromising mutation detection in patient samples and mimic mutation-induced alterations of mRNA.

Cellular Senescence↗
PubMed1982

RNA splicing in Neurospora mitochondria. Characterization of new nuclear mutants with defects in splicing the mitochondrial large rRNA.

In Neurospora, the gene encoding the mitochondrial large (25S) ribosomal RNA contains an intervening sequence of 2.3 kb. We have identified eight nuclear mutants that are defective in splicing the mitochondrial large ribosomal RNA and that accumulate unspliced precursor RNA. These mutants identify three different nuclear genes required for the same mitochondrial RNA splicing reaction. Some of the mutants have unique phenotypic characteristics (for example, accumulation of an unusual intron RNA) that may provide insight into specific aspects of mitochondrial RNA splicing. Mutations at one locus, cyt4, are subject to partial phenotypic suppression by the electron-transport inhibitor antimycin. This phenomenon suggests that at least one component required for mitochondrial RNA splicing is regulated such that its synthesis or activity is increased in response to impairment of electron transport.

Antimycin A↗
PubMed1982

A single-base change at a splice site in a beta 0-thalassemic gene causes abnormal RNA splicing.

We have studied the structure and transcription of a cloned human beta-globin gene from a fetus diagnosed for beta 0 thalassemia. The sequence of the beta 0 gene differs from that of a normal beta-globin gene at positions 1 and 74 of the second intervening sequence (IVS2). The position 1 change alters the GT dinucleotide conserved at 5' splice sites, while the position 74 change is a common sequence polymorphism. When the cloned beta 0 gene is introduced into HeLa cells by use of an SV40-derived plasmid vector, two abnormally spliced cytoplasmic beta-globin RNAs are detected. The predominant RNA differs from normal beta-globin mRNA by the insertion of the first 47 nucleotides of IVS2 between exons 2 and 3. The less abundant RNA comprises the normal first exon spliced directly to the third. Analysis of nuclear RNA suggests that the beta 0 transcript is inefficiently spliced and that the removal of the two intervening sequences is coupled.

Base Sequence↗
PubMed1984

RNA splicing in Neurospora mitochondria: nuclear mutants defective in both splicing and 3' end synthesis of the large rRNA.

We have identified nuclear mutants of Neurospora that are defective in splicing the mitochondrial large rRNA and that accumulate unspliced pre-rRNA (35S RNA). In cyt-4 mutants, the unspliced pre-rRNA contains short 3' end extensions (110 nucleotides) that are not present in pre-rRNAs from the other mutants. This and other characteristics suggest that the cyt-4 mutants may be primarily defective in 3' end synthesis and the RNA splicing defect occurs secondarily as a result of impaired RNA folding. The cyt-4 mutants also accumulate a "short" intron RNA and small exon RNAs that may reflect aberrant RNA cleavages. The 5' end of the short intron is about 285 nucleotides downstream from the 5' splice site at or near the base of the "central hairpin", a putative intermediate in folding of the pre-rRNA. Furthermore, the aberrant cleavage sites are immediately after a six nucleotide sequence (GAUAAU) homologous to the final splice junction (GAU/AAC).

Base Sequence↗
PubMed1986

One binding site determines sequence specificity of Tetrahymena pre-rRNA self-splicing, trans-splicing, and RNA enzyme activity.

The specificity of reactions catalyzed by the Tetrahymena pre-rRNA intervening sequence (IVS) was studied using site-specific mutagenesis. Two sequences required for 5' splice-site selection during self-splicing were defined. Single-base changes in either a 5' exon sequence or a 5' exon-binding site within the IVS disrupt their ability to pair and result in inefficient or inaccurate splicing. Combinations that restore complementarity suppress the effect of the single-base changes. Sequence alterations in the 5' exon-binding site also change the specificity of two other reactions: intermolecular exon ligation (trans-splicing) and the enzymatic nucleotidyltransferase activity of the IVS RNA. Thus the substrate specificity of an RNA enzyme can be changed in a manner predictable by the rules of Watson-Crick base-pairing.

Base Sequence↗
PubMed1987

Self-splicing of group II introns in vitro: lariat formation and 3' splice site selection in mutant RNAs.

Deletion or substitution of the branch A residue in group II intron bl1 significantly reduces splicing activity; yet, residual exon ligation is correct, and lariats have their branch points at the normal distance from the 3' end of the intron. Mutations in the sequence facing the branch point also allow residual lariat formation; however, free 3' exons are generated with false 5' termini, all of which are within a UCACA consensus sequence located upstream or downstream of the normal 3' splice site. These results indicate that both the conserved 3' splice site APy and the spatial arrangements in stem 6 are crucial for correct 3' splice site selection.

Electrophoresis, Polyacrylamide Gel↗
PubMed1987

5' cleavage site in eukaryotic pre-mRNA splicing is determined by the overall 5' splice region, not by the conserved 5' GU.

We have generated all possible single point mutations of the invariant 5' GT of the large beta-globin intron and determined their effect on splicing in vitro. None of the mutants prevented cleavage in the 5' splice region, but many reduced or abolished exon joining. The mutations GT----TT and GT----CT resulted in a shift of the 5' cleavage site on nucleotide upstream; in the case of the mutation GT----TT, this shift was reverted by a second site mutation within the 5' splice region. Our results suggest that the 5' cleavage site is determined not by the conserved GU sequence but by the 5' splice region as a whole, most probably via base-pairing to the 5' end of the U1 snRNA.

Animals↗
PubMed1988

Substrate recognition and splice site determination in yeast tRNA splicing.

S. cerevisae tRNA introns interrupt the gene at a constant position in the anticodon loop. Pre-tRNAs are matured by an endonuclease and a ligase. The endonuclease alone can accurately release the intron from the pre-tRNA. Here, we investigate the mechanism of splice site selection by the endonuclease. We propose that it initially recognizes features in the mature domain common to all tRNAs. Once positioned on the enzyme, the splice sites are recognizable because they are a fixed distance from the mature domain. To test this hypothesis, we developed a system for synthesizing pre-tRNA by bacteriophage T7 RNA polymerase. To search for recognition sites, we made several mutations. Mutations of C56 and U8 strongly affect endonuclease recognition of pre-tRNA. With insertion and deletion mutations, we show that the anticodon stem determines splicing specificity. The sequence and structure of the intron are not strong determinants of splice site selection.

Base Sequence↗