Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Protein Splicing”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 613 records · Page 34Linked to original sources
PubMed1999

The G protein beta3 subunit splice variant Gbeta3-s causes enhanced chemotaxis of human neutrophils in response to interleukin-8.

A C825T polymorphism was recently described in GNB3, the gene encoding the Gbeta3 subunit of heterotrimeric G proteins. The 825T allele is associated with the expression of a shorter splice variant (Gbeta3-s) and enhanced signal transduction via pertussis toxin (PTX)-sensitive G proteins. Given the pivotal role of G protein betagamma dimers in chemotaxis, we related the genotype at the GNB3 locus as a marker for Gbeta3-s expression to chemotaxis of human neutrophils in response to stimulation with interleukin-8 (IL-8). IL-8, which activates a CXC receptor coupled to PTX-sensitive G proteins, induced at 10 nM an enhanced maximum chemotaxis of neutrophils from individuals with TC/TT genotype compared to CC genotype. Furthermore, migration of neutrophils from 825T allele carriers was 2.5-fold higher at 0.1 nM and 1 nM IL-8. At these concentrations of IL-8, no significant chemotaxis was observed in neutrophils from homozygous C825 allele carriers, indicating a genotype-dependent, different potency of IL-8 to chemoattract neutrophils. In contrast, IL-8-induced Ca2+ signals and O2- generation were independent of genotype. The role of Gbeta3-s in enhanced chemotaxis could be confirmed by determination of chemotaxis of COS-7 cells following transfection with either Gbeta3-s or "wild-type" Gbeta3. Upon stimulation of the transfected cells with the chemoattractant lysophosphatidic acid (LPA), we observed an enhanced chemotactic response of Gbeta3-s-transfected compared to Gbeta3-transfected COS-7 cells, confirming that Gbeta3-s actually causes enhanced chemotaxis.

Alleles↗
PubMed1992

A tissue-specific small nuclear ribonucleoprotein and the regulated splicing of the calcitonin/calcitonin gene-related protein transcript.

Based on a correlation between expression patterns of an abundant tissue-specific small nuclear ribonucleoprotein (N protein) and the calcitonin gene-related protein (CGRP) splicing choice, a small nuclear ribonucleoprotein (the N factor) has been hypothesized to potentially function as a trans-acting factor involved in the regulation of the alternative splicing of the calcitonin/CGRP transcript. RNA analysis indicated that most rat, human, and simian cell lines and tissues making the CGRP mRNA splicing choice expressed the N factor mRNA. These data led us to address the effect of ectopic expression of the N factor in HeLa cells, which exhibit a calcitonin splicing choice when expressing the calcitonin/CGRP gene. Expression of the N factor exerts no effect on the calcitonin/CGRP splicing choice in HeLa cells. Furthermore, several cell lines such as the human 293 cell line make the CGRP mRNA splicing choice in the absence of any detectable level of the mRNA encoding the N factor. Together, these data reveal that the N protein is neither sufficient nor required for the tissue-specific CGRP splicing decision and that the N protein is not the trans-acting factor regulating the alternative splicing of the calcitonin/CGRP gene.

Animals↗
PubMed1984

Structure of mouse major urinary protein genes: different splicing configurations in the 3'-non-coding region.

The multigene family which codes for the mouse major urinary proteins (MUPs) consists of approximately 35 genes. Most of these are members of two different groups, Group 1 and Group 2, which can be distinguished by nucleic acid hybridisation. Here we describe the structure of a Group 1 gene and show that two size classes of MUP mRNA which are found in mouse liver result from different splicing events in the 3'-non-coding region and contain different polyadenylation sites. Short mRNA is approximately 750 nucleotides long, contains six exons, and is the main product of the Group 2 genes. Long mRNA is approximately 880 nucleotides long, contains seven exons and is the main product of the Group 1 genes. Five exons and part of the sixth are common to long and short mRNA and contain the coding region. This codes for an acidic protein of 180 amino acids containing an 18 residue signal peptide. A comparison of the mouse sequence with a homologous rat alpha 2u-globulin sequence shows that the rate of evolutionary divergence of the two proteins has been high. Silent sites have diverged four times more rapidly than replacement sites, showing that there has been selection against change in the protein sequence.

Amino Acid Sequence↗
PubMed2001

New roles for the Snp1 and Exo84 proteins in yeast pre-mRNA splicing.

The mammalian 70K protein, a component of the U1 small nuclear ribonucleoprotein involved in pre-mRNA splicing, interacts with a number of proteins important for regulating constitutive and alternative splicing. Similar proteins that interact with the yeast homolog of the 70K protein, Snp1p, have yet to be identified. We used the two-hybrid system to find four U1-Snp1 associating (Usa) proteins. Two of these proteins physically associate with Snp1p as assayed by coimmunoprecipitation. One is Prp8p, a known, essential spliceosomal component. This interaction suggests some novel functions for Snp1p and the U1 small nuclear ribonucleoprotein late in spliceosome development. The other, Exo84p, is a conserved subunit of the exocyst, an eight-protein complex functioning in secretion. We show here that Exo84p is also involved in pre-mRNA splicing. A temperature-sensitive exo84 mutation caused increased ratios of pre-mRNA to mRNA for the Rpl30 and actin transcripts in cells incubated at the non-permissive temperature. The mutation also led to a defect in splicing and prespliceosome formation in vitro; an indication that Exo84p has a direct role in splicing. The results elucidate a surprising link between splicing and secretion.

Actins↗
PubMed2001

Serine-arginine (SR) protein-like factors that antagonize authentic SR proteins and regulate alternative splicing.

We have characterized two RNA-binding proteins, of apparent molecular masses of approximately 40 and 35 kDa, which possess a single N-terminal RNA-recognition motif (RRM) followed by a C-terminal domain rich in serine-arginine dipeptides. Their primary structures resemble the single-RRM serine-arginine (SR) protein, SC35; however their functional effects are quite distinctive. The 40-kDa protein cannot complement SR protein-deficient HeLa cell S100 extract and showed a dominant negative effect in vitro against the authentic SR proteins, SF2/ASF and SC35. Interestingly, the 40- and 35-kDa proteins antagonize SR proteins and activate the most distal alternative 5' splice site of adenovirus E1A pre-mRNA in vivo, an activity that is similar to that characterized previously for the heterogeneous nuclear ribonucleoprotein particles A/B group of proteins. A series of recombinant chimeric proteins consisting of domains from these proteins and SC35 in various combinations showed that the RRM, but not the C-terminal domain rich in serine-arginine dipeptides, has a dominant role in this activity. Because of the similarity to SR proteins we have named these proteins SRrp40 and SRrp35, respectively, for SR-repressor proteins of approximately 40 and approximately 35 kDa. Both factors show tissue- and cell type-specific patterns of expression. We propose that these two proteins are SR protein-like alternative splicing regulators that antagonize authentic SR proteins in the modulation of alternative 5' splice site choice.

3T3 Cells↗
PubMed1990

PRP5: a helicase-like protein required for mRNA splicing in yeast.

A 96-kDa protein predicted by the DNA sequence of the Saccharomyces cerevisiae PRP5 gene contains a domain that bears a striking resemblance to a family of RNA helicases characterized by the conserved amino acid sequence Asp-Glu-Ala-Asp (D-E-A-D). Previous work indicated that the product of the PRP5 gene is required for splicing and that spliceosome assembly does not occur in its absence. However, its precise role in splicing and the nature of its biochemical activity remained unknown. To examine the role of PRP5 in splicing, we cloned the gene by complementation of a temperature-sensitive mutation and determined its DNA sequence. We discuss here the possible roles for an RNA helicase in splicing and for the activity of the PRP5 protein.

Amino Acid Sequence↗
PubMed1998

Characterization of the gene encoding the human Kidd blood group/urea transporter protein. Evidence for splice site mutations in Jknull individuals.

The Kidd (JK) blood group is carried by an integral membrane glycoprotein which transports urea through the red cell membrane and is also present on endothelial cells of the vasa recta in the kidney. The exon-intron structure of the human blood group Kidd/urea transporter gene has been determined. It is organized into 11 exons distributed over 30 kilobase pairs. The mature protein is encoded by exons 4-11. The transcription initiation site was identified by 5'-rapid amplification of cDNA ends-polymerase chain reaction at 335 base pairs upstream of the translation start point located in exon 4. The 5'-flanking region, from nucleotide -837 to -336, contains TATA and inverted CAAT boxes as well as GATA-1/SP1 erythroid-specific cis-acting regulatory elements. Analysis of the 3'-untranslated region reveals that the two equally abundant erythroid transcripts of 4.4 and 2.0 kilobase pairs arise from usage of different alternative polyadenylation signals. No obvious abnormality of the Kidd/urea transporter gene, including the 5'- and 3'-untranslated regions, has been detected by Southern blot analysis of the blood of two unrelated Jknull individuals (B.S. and L.P.), which lacks all Jk antigens and Jk proteins on red cells, but was genotyped as homozygous for a "silent" Jkb allele. Further analysis indicated that different splice site mutations occurred in each variant. The first mutation affected the invariant G residue of the 3'-acceptor splice site of intron 5 (variant B.S.), while the second mutation affected the invariant G residue of the 5'-donor splice site of intron 7 (variant L.P.). These mutations caused the skipping of exon 6 and 7, respectively, as seen by sequence analysis of the Jk transcripts present in reticulocytes. Expression studies in Xenopus oocytes demonstrated that the truncated proteins encoded by the spliced transcripts did not mediate a facilitated urea transport compared with the wild type Kidd/urea transporter protein and were not expressed on the oocyte's plasma membrane. These findings provide a rational explanation for the lack of Kidd/urea transporter protein and defect in urea transport of Jknull cells.

Base Sequence↗
PubMed2001

RNA splicing at human immunodeficiency virus type 1 3' splice site A2 is regulated by binding of hnRNP A/B proteins to an exonic splicing silencer element.

The synthesis of human immunodeficiency virus type 1 (HIV-1) mRNAs is a complex process by which more than 30 different mRNA species are produced by alternative splicing of a single primary RNA transcript. HIV-1 splice sites are used with significantly different efficiencies, resulting in different levels of mRNA species in infected cells. Splicing of Tat mRNA, which is present at relatively low levels in infected cells, is repressed by the presence of exonic splicing silencers (ESS) within the two tat coding exons (ESS2 and ESS3). These ESS elements contain the consensus sequence PyUAG. Here we show that the efficiency of splicing at 3' splice site A2, which is used to generate Vpr mRNA, is also regulated by the presence of an ESS (ESSV), which has sequence homology to ESS2 and ESS3. Mutagenesis of the three PyUAG motifs within ESSV increases splicing at splice site A2, resulting in increased Vpr mRNA levels and reduced skipping of the noncoding exon flanked by A2 and D3. The increase in Vpr mRNA levels and the reduced skipping also occur when splice site D3 is mutated toward the consensus sequence. By in vitro splicing assays, we show that ESSV represses splicing when placed downstream of a heterologous splice site. A1, A1(B), A2, and B1 hnRNPs preferentially bind to ESSV RNA compared to ESSV mutant RNA. Each of these proteins, when added back to HeLa cell nuclear extracts depleted of ESSV-binding factors, is able to restore splicing repression. The results suggest that coordinate repression of HIV-1 RNA splicing is mediated by members of the hnRNP A/B protein family.

3' Untranslated Regions↗
PubMed1998

Oncoprotein TLS interacts with serine-arginine proteins involved in RNA splicing.

The gene encoding the human TLS protein, also termed FUS, is located at the site of chromosomal translocations in human leukemias and sarcomas where it forms a chimeric fusion gene with one of several different genes. To identify interacting partners of TLS, we screened a yeast two-hybrid cDNA library constructed from mouse hematopoietic cells using the C-terminal region of TLS in the bait plasmid. Two cDNAs encoding members of the serine-arginine (SR) family of proteins were isolated. The first SR protein is the mouse homolog of human splicing factor SC35, and the second SR member is a novel 183-amino acid protein that we term TASR (TLS-associated serine-arginine protein). cDNA cloning of human TASR indicated that mouse and human TASR have identical amino acid sequences. The interactions between TLS and these two SR proteins were confirmed by co-transfection and immunoprecipitation studies. In vivo splicing assays indicated that SC35 and TASR influence splice site selection of adenovirus E1A pre-mRNA. TLS may recruit SR splicing factors to specific target genes through interaction with its C-terminal region, and chromosomal translocations that truncate the C-terminal region of TLS may prevent this interaction. Thus TLS translocations may alter RNA processing and play a role in malignant transformation.

Amino Acid Sequence↗
PubMed1997

In vivo regulation of alternative pre-mRNA splicing by the Clk1 protein kinase.

Controlled expression of cellular and viral genes through alternative precursor messenger RNA (pre-mRNA) splicing requires serine/arginine-rich (SR) proteins. The Clk1 kinase, which phosphorylates SR proteins, is regulated through alternative splicing of the Clk1 pre-mRNA, yielding mRNAs encoding catalytically active and truncated inactive polypeptides (Clk1 and Clk1T, respectively). We present evidence that Clk1 and Clk1T proteins regulate the splicing of Clk1 and adenovirus pre-mRNAs in vivo. The peptide domain encoded by the alternatively spliced exon of Clk1 is essential for the regulatory activity of the Clk1 kinase. This is the first direct demonstration of an in vivo link between alternative splicing and protein kinase activity.

Adenovirus E1A Proteins↗
PubMed2003

Mouse ADAM33: two splice variants differ in protein maturation and localization.

We compared the tissue mRNA prevalence and protein maturation of two splice variants of mouse ADAM33, a metalloprotease implicated in airway hyperresponsiveness. These variant cDNAs, designated 914 (alpha) and 906 (beta), encode membrane-bound forms that differ primarily in 26 residues (exon 17) between the cysteine-rich and epidermal growth factor-like domains. Proteins of approximately 120 and 103 kD, detectable by anti-ADAM33 antibodies, were expressed in 914-transfected HEK293 cells. The time-dependent appearance of the approximately 100-kD form and its inhibition by a peptidyl chloromethylketone, or the calcium ionophore, A23187, indicated that this was mature ADAM33, which was processed by a furin-like convertase. One form, approximately 110 kD, was detected in 906-transfected cell lysates. Trypsin and biotinylation treatment of transfected cells demonstrated that all of the mature approximately 100-kD, a minority of the approximately 120-kD pro-form, and none of the 906-expressed 110-kD form localized to the cell surface. The mature form was resistant to endoglycosidase H(f). The approximately 110-kD form was endoglycosidase H(f)-sensitive, indicating retention proximal to the trans-Golgi, consistent with a lack of maturation. Quantitation of transcripts demonstrated that those containing exon 17 predominate, whereas those lacking exon 17 are negligible in the mouse lung, although detectable at low levels in mouse testis, heart, and brain. Thus, potential dominant-negative effects exerted by the nonprocessed 906-encoded beta splice variant are unlikely to occur in mouse lung.

ADAM Proteins↗
PubMed1993

Structure of the murine Mac-2 gene. Splice variants encode proteins lacking functional signal peptides.

The murine Mac-2 gene is composed of six exons dispersed over 10.5 kilobases. S1 nuclease mapping showed multiple transcription initiation sites, clustered within a 30-base pair region. Sequence analysis revealed that a consensus initiator sequence is located in this area which lacks a TATA motif. The untranslated first exon contains an alternative splice donor site, confirming the existence of two cDNA species with the potential to encode proteins differing at their NH2 termini. In vitro expression and translocation experiments demonstrate that both of the alternatively spliced variants of Mac-2 encode proteins which lack a functional signal peptide. Subcellular fractionation studies indicate that most of the Mac-2 protein is present in the cytosol. These results support the view that Mac-2 is exported from the cell by an unusual mechanism which does not depend on the presence of a signal peptide.

Alternative Splicing↗
PubMed1993

Interactions between PRP9 and SPP91 splicing factors identify a protein complex required in prespliceosome assembly.

The PRP9 protein is a yeast splicing factor implicated in the early steps of spliceosome assembly whose sequence contains an amino-terminal putative leucine zipper structure and two carboxy-terminal motifs reminiscent of zinc fingers. Here, we show that the deletion of the second carboxy-terminal motif results in a dominant lethal phenotype. This observation, combined with an in vivo-binding assay for protein-protein interactions, reveals the presence of two distinct binding sites on the PRP9 protein. The carboxy-terminal region contributes to the PRP9 homodimerization, whereas the amino-terminal region binds the SPP91 splicing factor. Further experiments suggest that other factors bind to PRP9 and SPP91 proteins. Finally, we demonstrate that the PRP9 protein acts after the formation of the U1 snRNP-pre-mRNA complex. The existence of a protein complex including the PRP9 factor is discussed.

Binding Sites↗
PubMed1993

Genomic organization of the human HBNF gene and characterization of an HBNF variant protein as a splice mutant.

The organization of the human heparin-binding neurite outgrowth promoting factor (HBNF) gene is presented. Based on Southern analysis and the isolation of genomic DNA clones from a lambda phage library, the minimum size of the gene is 42 kb. Sequences comprising the HBNF mRNA are contained in five exons which account for the 1650 nt mRNA size observed by northern analysis. From the structure of the gene it is predicted that a variant human HBNF cDNA with a three basepair deletion is a result of alternative splicing at the acceptor site of exon 5. Evidence is presented that indicates the existence of a variant HBNF protein, des-Ala119-HBNF, in bovine brain which has a corresponding amino acid deletion. This alternate form comprises approximately 20% of the total HBNF protein present in bovine brain.

Alternative Splicing↗
PubMed1997

A truncated isoform of Ca2+/calmodulin-dependent protein kinase II expressed in human islets of Langerhans may result from trans-splicing.

Calcium/calmodulin-dependent protein kinase II (CaM kinase II) has been proposed to play a key role in glucose stimulated insulin secretion. Using the rapid amplification of cDNA ends technique we amplified the 3' end of the CaM kinase II gamma gene from human islet RNA. A novel cDNA was detected composed of 5' sequence from the human CaM kinase II gamma gene joined to the 3' end of the human signal recognition particle 72 (SRP72) gene. We predict that this mRNA species will code for a truncated form of CaM kinase II, designated gammaSRP, comprising the entire catalytic and regulatory domains of the protein and with a predicted molecular weight of 37 kDa. We mapped the human SRP72 gene to chromosome 18 and, as the CaM kinase II gamma gene was previously mapped to human chromosome 10q22, we suggest this novel cDNA may have resulted from trans-splicing.

Alternative Splicing↗
PubMed1994

The Drosophila sex determination gene snf encodes a nuclear protein with sequence and functional similarity to the mammalian U1A snRNP protein.

Alternative splicing controls the expression of many genes, including the Drosophila sex determination gene Sex-lethal. Previous studies have suggested that snf plays a role in regulating Sex-lethal splicing. Here, we demonstrate that snf is an integral component of the machinery required for splice site recognition. We have cloned snf and found that it has sequence homology to the mammalian U1A and U2B" snRNP proteins. Moreover, we establish that snf encodes a Drosophila protein shown previously to have functional similarity to U1A. Finally, with the isolation and analysis of a null mutation, we demonstrate that snf is an essential gene. These studies provide the first demonstration, in a multicellular organism, that mutations in a U1 snRNP protein alter splicing in vivo.

Alleles↗
PubMed1987

Aberrant splicing of proteolipid protein mRNA in the dysmyelinating jimpy mutant mouse.

cDNA clones encoding proteolipid protein (PLP) were isolated from a mouse brain library and sequenced. We describe two transcripts arising from the PLP locus by alternative splicing: the major one encodes the 277-amino acid PLP protein and the minor one corresponds to the DM-20 protein, a PLP-like protein of 20,000 Mr that shares both amino and carboxyl regions with PLP. These two transcripts lack approximately 70 bases in PLP mRNA from the dysmyelinating jimpy mutant. The deletion spans amino acids 208-232; however, this region is present in the jimpy PLP-encoding gene. We propose that the jimpy mutant suffers a point mutation or the deletion of a few bases in the PLP gene that alters the normal splicing pattern and generates partially deleted PLP transcripts.

Amino Acid 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↗