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PubMed2003

Genomic organization of human myotonic dystrophy kinase-related Cdc42-binding kinase alpha reveals multiple alternative splicing and functional diversity.

Myotonic dystrophy kinase-related Cdc42-binding kinase alpha (MRCKalpha) is a Cdc42/Rac interactive binding-containing serine/threonine kinase with multiple functional domains. Its roles in the regulation of peripheral actin reorganization in HeLa cells and NGF-induced neurite outgrowth in PC12 cells have been documented. Here we report the characterization of the genomic structure and alternative splicing of the human counterpart. Human MRCKalpha gene is located on chromosome 1q42.1, spanning a genomic region of 250-300 kb and is composed of 41 exons. Four exons in the internal variable region and six in the 3' end were found to undergo extensive alternative splicing, giving rise to 96 possible transcripts of different combinations. The region of the internal splice site that defines a variable region in between two functional domains of opposite regulatory effects on MRCKalpha catalytic activity, and the 3' end splice site that generates variants with differential GTPase binding activity suggest a role for these alternative splicing events in MRCKalpha regulation.

3' Untranslated Regions↗

PubMed2004

Genomic organisation of the human MDM2 oncogene and relationship to its alternatively spliced mRNAs.

The MDM2 proto-oncogene, which encodes a protein that binds to the p53 tumour suppressor, has been found amplified and overexpressed in a range of human tumours. Although the human MDM2 cDNA sequence has been reported, the genomic organisation of the human gene has not been documented. We have previously reported the detection of five alternative internally deleted MDM2 transcripts in human tumours and suggested these may represent alternatively spliced forms. Here we demonstrate two novel MDM2 transcripts with internal deletions, using RT-PCR followed by sequencing. To definitively ascribe these variant transcript forms to alternative splicing, and to explore associated mechanisms, we have determined the intron--exon organisation of the human genomic sequence. The human MDM2 gene spans approximately 33 kb and is divided into 12 exons. Exon sizes range from 50 to > or =1161 bp and intron sizes vary from 121 to approximately 7000 bp. The positions of intron--exon boundaries are compared with the deletion junctions of the multiple-sized transcripts and discussed in relation to alternative splicing mechanism.

Alternative Splicing↗

PubMed1994

Antigenic determinants encoded by alternatively spliced exons of CD45 are determined by the polypeptide but influenced by glycosylation.

Antibodies recognising the products of alternatively spliced exons near the N-terminus of the leukocyte common antigen, CD45, have been widely used to distinguish populations of lymphocytes with different functional properties. These alternatively spliced regions contain a high content of serine and threonine residues (average 35%) and are heavily O-glycosylated. Despite evidence that the O-glycosylation contributes significantly to the antigenic character of this region of CD45, work with leukosialin and mucin glycoproteins leads to the prediction that the majority of epitopes in the N-terminal exons should be linear protein determinants. In this study the exons of CD45 were expressed in Escherichia coli as non-glycosylated proteins fused to glutathione S-transferase (GST). Fourteen out of 17 mAbs specific for human CD45R reacted with a fusion protein containing exons 4, 5 and 6 (ABC) of human CD45, and four out of six mAbs specific for rat CD45R reacted with an equivalent rat protein. mAbs recognising the product of rat exon B are reported for the first time. Kinetic analysis of MRC OX22 antibody binding to spleen CD45 and to GST fusion proteins showed that the carbohydrate affected the kinetics of binding of antibodies to the protein backbone. In conclusion, heterogeneity in the glycosylation of heavily O-glycosylated cell surface proteins can affect interactions of these proteins both directly through the carbohydrate and indirectly through effects on the protein backbone.

Alternative Splicing↗

PubMed1991

Developmentally regulated alternative splicing of Drosophila myosin heavy chain transcripts: in vivo analysis of an unusual 3' splice site.

The 3' penultimate exon (exon 18) of transcripts from the muscle myosin heavy chain (MHC) gene of Drosophila melanogaster is excluded from mRNAs of embryonic and larval muscle, while it is included in mRNAs of adult thoracic muscles. By transforming organisms with the MHC gene 5' end, linked in-frame to the MHC gene 3' end, we were able to generate correct tissue-specific expression of this minigene and stage-specific splicing of exon 18, indicating that all the cis-acting sequences necessary for alternative splicing are contained within the construct. The 3' splice site that precedes exon 18 is unusually purine-rich, may form a stem-loop structure with the 5' splice site following exon 18, and is conserved relative to the splice site of an alternative exon of the Drosophila alkali myosin light chain gene. We converted the MHC gene 3' splice junction to a consensus splice site and also inserted the branchpoint and 3' splice site of a constitutively spliced intron in its place. These alterations had no effect on the splicing pathway in vivo, ruling out the possibility that the unusual splice junction, or secondary structures that involve this splice junction, directly regulate alternative splicing of exon 18.

Animals↗

PubMed2002

Identification of a cis element for tissue-specific alternative splicing of chloroplast ascorbate peroxidase pre-mRNA in higher plants.

Alternative splicing events in the 3'-terminal region of chloroplast ascorbate peroxidase (chlAPX) pre-mRNA in spinach and tobacco, which produced four types of mRNA variants, one form (tAPX-I) encoding thylakoid-bound APX (tAPX) and three forms (sAPX-I, -II, and -III) encoding stromal APX (sAPX), were regulated in a tissue-specific manner. The ratio of the level of sAPX mRNAs (sAPX-I, -II, and -III) to tAPX-I mRNA was close to 1 in leaf, whereas the ratio in root was greatly elevated due to an increase in sAPX-III and a decrease in tAPX-I resulting from the alternative excision of intron 11 and intron 12, respectively. A putative splicing regulatory cis element (SRE), which is highly conserved in the sequences of chlAPX genes of higher plants, was identified upstream of the acceptor site in intron 12. The deletion of the SRE sequence diminished the splicing efficiency of intron 12 in tobacco leaf in vivo. Gel-shift analysis showed that SRE interacts strongly with a nuclear protein from leaves but not those from the roots of spinach and tobacco. These results indicate that the tissue-specific alternative splicing of chlAPX pre-mRNA is regulated by the splicing enhancer SRE.

Alternative Splicing↗

PubMed1995

Identification of the alternative spliced form of the alpha 2/delta subunit of voltage sensitive Ca2+ channels expressed in PC12 cells.

The alpha 2/delta subunit of voltage sensitive Ca2+ channels expressed in PC12 has been cloned and partially sequenced. The message observed in Northern blot analysis displays a 7.5 kb transcript, identical in size to mRNA of rabbit skeletal muscle and rat brain. The nucleotide sequence of the cloned alpha 2 subunit of the PC12 specific cDNA is > 99% identical to rat brain sequence and 85% to skeletal muscle. Reverse-transcriptase-polymerase chain reaction (RT-PCR) of the alternative splicing region identifies two deleted regions of 57 bp and 21 bp in PC12 expressed alpha 2/delta transcript. The alternative variant alpha 2e of alpha 2/delta subunit which is expressed in PC12 cells was previously identified in human embryonic kidney (HEK293) cells. RT-PCR analysis show two different sized alternative PCR fragments in rat lung and none in rat spleen, kidney and intestine. Antibodies prepared against a 19 amino acid peptide within the alternative spliced region effectively inhibits [3H]dopamine release in PC12 cells. This implies that the alternatively spliced region is positioned extracellularly and is involved in regulation of the L-type Ca2+ channel-mediated transmitter release.

Alternative Splicing↗

PubMed2004

Lipopolysaccharide-induced expression of multiple alternatively spliced MEFV transcripts in human synovial fibroblasts: a prominent splice isoform lacks the C-terminal domain that is highly mutated in familial Mediterranean fever.

OBJECTIVE: To investigate the expression of the familial Mediterranean fever (FMF) gene (MEFV) in human synovial fibroblasts. METHODS: MEFV messenger RNA in synovial fibroblasts, chondrocytes, and peripheral blood leukocytes (PBLs) was analyzed by semiquantitative and real-time polymerase chain reaction and ribonuclease protection assay. The subcellular localization of pyrin, the MEFV product, was determined in transfected synovial fibroblasts and HeLa cells with plasmids encoding pyrin isoforms. Native pyrin was detected with an antipyrin antibody. RESULTS: MEFV was expressed in synovial fibroblasts, but not in chondrocytes. Four alternatively spliced transcripts were identified: an extension of exon 8 (exon 8ext) resulting in a frameshift that predicts a truncated protein lacking exons 9 and 10, the addition of an exon (exon 4a) predicting a truncated protein at exon 5, the in-frame substitution of exon 2a for exon 2, and the previously described removal of exon 2 (exon 2Delta). Exon 8ext transcripts represented 27% of the total message population in synovial fibroblasts. All other alternatively spliced transcripts were rare. Consensus and alternatively spliced transcripts were induced by lipopolysaccharide in synovial fibroblasts and PBLs. In transfected cells, the proteins encoded by all highly expressed splice forms were cytoplasmic. In contrast, native pyrin was predominantly nuclear in synovial fibroblasts, neutrophils, and dendritic cells, but was cytoplasmic in monocytes. CONCLUSION: Several MEFV transcripts are expressed and inducible in synovial fibroblasts. A prominent isoform lacks the C-terminal domain that contains the majority of mutations found in patients with FMF. While recombinant forms of all major pyrin isoforms are cytoplasmic, native pyrin is nuclear in several cell types. Thus, mechanisms in addition to splicing patterns must control pyrin's subcellular distribution.

Alternative Splicing↗

PubMed2006

HOLLYWOOD: a comparative relational database of alternative splicing.

RNA splicing is an essential step in gene expression, and is often variable, giving rise to multiple alternatively spliced mRNA and protein isoforms from a single gene locus. The design of effective databases to support experimental and computational investigations of alternative splicing (AS) is a significant challenge. In an effort to integrate accurate exon and splice site annotation with current knowledge about splicing regulatory elements and predicted AS events, and to link information about the splicing of orthologous genes in different species, we have developed the Hollywood system. This database was built upon genomic annotation of splicing patterns of known genes derived from spliced alignment of complementary DNAs (cDNAs) and expressed sequence tags, and links features such as splice site sequence and strength, exonic splicing enhancers and silencers, conserved and non-conserved patterns of splicing, and cDNA library information for inferred alternative exons. Hollywood was implemented as a relational database and currently contains comprehensive information for human and mouse. It is accompanied by a web query tool that allows searches for sets of exons with specific splicing characteristics or splicing regulatory element composition, or gives a graphical or sequence-level summary of splicing patterns for a specific gene. A streamlined graphical representation of gene splicing patterns is provided, and these patterns can alternatively be layered onto existing information in the UCSC Genome Browser. The database is accessible at http://hollywood.mit.edu.

Alternative Splicing↗

PubMed1998

Expressional changes in alternative splicing affecting genes during cell passage of human diploid fibroblasts.

Normal human diploid cells have a limited proliferative lifespan in in vitro cultures. Changes in gene expression have been examined for understanding control mechanisms of limited proliferative lifespan. and enhanced expression of growth suppressing genes such as p21 was reported in late-passaged cells. We screened genes which were expressed preferentially in mid-passaged cells by the differential plaque screening of the subtracted cDNA libraries prepared from young, life-extended, and immortalized SV40-transformed human fibroblasts. Among isolated clones, ASF/SF2, which was known to affect alternative splicing, was expressed in normal fibroblasts with a peak at mid-passage. Relative expression levels of SC35 and hnRNPA1, which are also known to affect alternative splicing, was also highest at mid-passage. Changes in alternative splicing at mid-passage, if it occurred, may play a crucial role in the process of cellular senescence.

Alternative Splicing↗

PubMed1996

Alternatively spliced forms in the cytoplasmic domain of the human growth hormone (GH) receptor regulate its ability to generate a soluble GH-binding protein.

The mechanism underlying the generation of soluble growth hormone binding protein (GHBP) probably differs among species. In rats and mice, it involves an alternatively spliced mRNA, whereas in rabbits, it involves limited proteolysis of the membrane-bound growth hormone receptor (GHR). In humans, this latter mechanism is favored, as no transcript coding for a soluble GHR has been detected so far. To test this hypothesis, we analyzed COS-7 cells transiently expressing the full-length human (h) GHR and observed specific GH-binding activity in the cell supernatants. Concomitantly, an alternatively spliced form in the cytoplasmic domain of GHR, hGHR-tr, was isolated from several human tissues. hGHR-tr is identical in sequence to hGHR, except for a 26-bp deletion leading to a stop codon at position 280, thereby truncating 97.5% of the intracellular domain of the receptor protein. When compared with hGHR, hGHR-tr showed a significantly increased capacity to generate a soluble GHBP. Interestingly, this alternative transcript is also expressed in liver from rabbits, mice, and rats, suggesting that, in these four species, proteolysis of the corresponding truncated transmembrane GHR is a common mechanism leading to GHBP generation. These findings support the hypothesis that GHBP may at least partly result from alternative splicing of the region encoding the intracellular domain and that the absence of a cytoplasmic domain may be involved in increased release of GHBP.

Alternative Splicing↗

PubMed1992

Ankyrin regulation: an alternatively spliced segment of the regulatory domain functions as an intramolecular modulator.

This study of two forms of ankyrin (protein 2.1 and 2.2) from human erythrocytes has revealed a role for alternate exon usage at the level of regulation of protein interactions. The smaller form of ankyrin (protein 2.2), which lacks a portion of the regulatory domain due to alternative splicing of pre-mRNA, exhibits increased affinity for the cytoplasmic domain of the anion exchanger, spectrin, and tubulin. Direct evidence that at least one of these associations is modulated by the alternatively spliced segment of the regulatory domain is provided by experiments utilizing a polypeptide that is comprised of residues 1513-1674 corresponding to the portion of the regulatory domain missing from protein 2.2. Addition of this regulatory domain polypeptide to binding assays reversed the increase in affinity of protein 2.2 for the anion exchanger. The inhibitory activity of the regulatory domain polypeptide in these assays is accompanied by a direct interaction with a site that is available on the smaller form of ankyrin and is distinct from the binding site for the anion exchanger. These results support the idea that the alternatively spliced segment within the regulatory domain of erythrocyte ankyrin performs a repressor function and acts through an allosteric mechanism involving interaction(s) at a site separate from the binding site for the anion exchanger.

Allosteric Regulation↗

PubMed2004

Expression and function of Slit1alpha, a novel alternative splicing product for slit1.

Slits are large molecular and extracellular glycoproteins that may function as chemorepellents in axon guidance and neural cell migration. The heterogeneity of the mRNA for slit has been described. Its variants indicate considerable potential for alternative splicing, resulting in the generation of multiple protein isoforms. We examined the regions in which these isoforms are expressed, and identified the highest expression of a splicing product for slit1 in rat brain rather than in other organs. The splicing product, Slit1alpha, arises through alternative splicing at the C-terminus of Slit1, causing defects in the cysteine knot domain. We show that slit1alpha exists in the hippocampus and cerebral cortex in rat brain by in situ hybridization, and that it acts as a chemorepellent in olfactory bulb axon guidance in vitro. These findings suggest that Slit1alpha is an active Slit1 protein specific in the vertebrate nervous system.

Alternative Splicing↗

PubMed1999

Goldfish brain GluR2: multiple forms, RNA editing, and alternative splicing.

cDNA coding for a full-length goldfish alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor subunit, GluR2, was cloned by screening unidirectional and bidirectional goldfish brain cDNA libraries. The clone has an open reading frame of 2679 bp, encoding a protein of 893 amino acids. Partial cDNA clones for three other GluR2 subunits were identified. GluR2 from goldfish brain exhibits RNA editing and alternative splicing. RNA editing occurred at the two sites demonstrated for mammalian GluR2 (Q/R and R/G). Unlike rat GluR2, GFGluR2a has a long (68 amino acids) C-terminal tail. Analysis of genomic DNA suggests that an alternatively spliced shorter C-terminal tail can be produced, similar to the rat protein. Thus, in goldfish brain, GluR2 exhibits diversity arising from multiple subtypes, RNA editing, and alternative splicing.

Alternative Splicing↗

PubMed1995

Adhesion activity in fibronectin's alternatively spliced domain EDa (EIIIA): complementarity to plasma fibronectin functions.

The EDa (EIIIA) domain is one of two alternatively spliced type III homology repeats in fibronectins which differentiate cellular (cFN) from plasma isoforms (pFN). A bacteria-expressed recombinant polypeptide encoding the EDa type III repeat promotes adhesion of some cell types and its activity is synergistic with neighboring repeats III11 and III12. We show in this study that co-coating substrata with the EDa-only polypeptide and a suboptimal concentration of pFN leads to increased attachment and extensive spreading of v-src-transformed 3T3 cells relative to that found on substrata of suboptimal pFN or EDa polypeptide alone. This complementarity of activities requires as little as 1 microgram/ml EDa polypeptide in the adsorbing mixture and displays sequence specificity for only EDa (recombinant polypeptides of neighboring repeats III11 or III12 were without effect). Furthermore, stress fibers and focal contacts are inducible on the EDa:pFN mixture, suggesting that the EDa sequence and its receptor participate in signal transduction. The codistribution of phosphotyrosine proteins, including pp125FAK, along with vinculin and talin into focal contacts supports this hypothesis. Therefore, an alternatively spliced domain EDa which is expressed in various proportions in cells and tissues may have special functions related to adhesion processes by complementing the functions of pFN circulating in blood.

3T3 Cells↗

PubMed2005

Identification and analysis of alternative splicing events conserved in human and mouse.

Alternative pre-mRNA splicing affects a majority of human genes and plays important roles in development and disease. Alternative splicing (AS) events conserved since the divergence of human and mouse are likely of primary biological importance, but relatively few of such events are known. Here we describe sequence features that distinguish exons subject to evolutionarily conserved AS, which we call alternative conserved exons (ACEs), from other orthologous human/mouse exons and integrate these features into an exon classification algorithm, acescan. Genome-wide analysis of annotated orthologous human-mouse exon pairs identified approximately 2,000 predicted ACEs. Alternative splicing was verified in both human and mouse tissues by using an RT-PCR-sequencing protocol for 21 of 30 (70%) predicted ACEs tested, supporting the validity of a majority of acescan predictions. By contrast, AS was observed in mouse tissues for only 2 of 15 (13%) tested exons that had EST or cDNA evidence of AS in human but were not predicted ACEs, and AS was never observed for 11 negative control exons in human or mouse tissues. Predicted ACEs were much more likely to preserve the reading frame and less likely to disrupt protein domains than other AS events and were enriched in genes expressed in the brain and in genes involved in transcriptional regulation, RNA processing, and development. Our results also imply that the vast majority of AS events represented in the human EST database are not conserved in mouse.

Alternative Splicing↗

PubMed2006

Alternative splicing of the voltage-gated Ca2+ channel beta4 subunit creates a uniquely folded N-terminal protein binding domain with cell-specific expression in the cerebellar cortex.

Ca2+ channel beta subunits regulate cell-surface expression and gating of voltage-dependent Ca2+ channel alpha1 subunits. Based on primary sequence comparisons, beta subunits are predicted to be modular structures composed of five domains (A-E) that are related to the large family of membrane-associated guanylate kinase proteins. The crystal structure of the beta subunit core B-D domains has been reported recently; however, little is known about the structures of the A and E domains. The N-terminal A domain differs among the four subtypes of Ca2+ channel beta subunits (beta1-beta4) primarily as the result of two duplications of an ancestral gene containing multiple alternatively spliced exons. At least nine A domain sequences can be generated by alternative splicing. In this report, we focus on one A domain sequence, the highly conserved beta4a A domain. We solved its three-dimensional structure and show that it is expressed in punctate structures throughout the molecular layer of the cerebellar cortex. We also demonstrate that it does not participate directly in Cav2.1 Ca2+ channel gating but serves as a binding site in protein-protein interactions with synaptotagmin I and the LC2 domain of microtubule-associated protein 1A. With respect to beta4 subunits, the interactions are specific for the beta4a splice variant, because they do not occur with the beta4b A domain. These results have strong bearing on our current understanding of the structure of alternatively spliced Ca2+ channel beta subunits and the cell-specific roles they play in the CNS.

Alternative Splicing↗

PubMed2003

Alternatively spliced, truncated human BRCA2 isoforms contain a novel coding exon.

The protein truncation test (PTT) employs in vitro transcription and translation of amplified cDNA and exonic gDNA to reveal truncating germ-line mutations. In a series of PTT analyses, abnormal splicing in the region encompassing exons 20-23 of BRCA2 was discovered in leucocytes from high-risk breast cancer patients. Although sequencing of the genomic DNA in this region failed to reveal a detectable mutation in these patients, cDNA obtained from this region of BRCA2 uncovered numerous alternative splice isoforms. PTT analysis and nested RT-PCR using RNA from leucocytes from healthy individuals, normal tissue and breast and ovarian cancer tumours demonstrated the presence of these alternatively spliced transcripts in all cases. The splice forms appeared to be more prominent in RNA from aged blood, suggesting that isoform expression was conditional. It is therefore important to distinguish naturally occurring alternative splicing from true splice defects due to mutations when interpreting PTT results.

Alternative Splicing↗

PubMed1991

Alternative splicing in the neural cell adhesion molecule pre-mRNA: regulation of exon 18 skipping depends on the 5'-splice site.

Two isoforms of the neural cell adhesion molecule (NCAM), termed NCAM-180 and NCAM-140, derive from a single gene via inclusion or exclusion of the penultimate exon 18 (E18). This alternative splicing event is tissue-specific and regulated during differentiation. To explore its structural basis, we have analyzed the pattern of spliced mRNA generated from transiently transfected minigenes construct containing this exon and portions of the adjacent introns and exons faithfully reproduces the differentiation state-dependent alternative splicing of the endogenous pre-mRNA. By systematic deletion and replacement analysis, we scanned the minigene for the presence of functionally important cis-elements. We identified two sequences that affected differentiation state-dependent regulation. One, the central part of E18, does not seem to contain a specific cis-element essential for proper splice site choice, because extending the deletion restored correctly regulated expression of the splicing products. In contrast, the 5'-splice site is an important element for regulation. Replacing it with a corresponding sequence from the alpha-globin gene resulted in constitutive use of the optional exon. When placed in the alpha-globin gene it did not promote alternative splicing. Instead, we observed a strongly decreased efficiency of splicing of the downstream intron in undifferentiated cells. This block of splicing was partially relieved after differentiation. The results are consistent with a model in which skipping of E18 is controlled in part at the associated 5'-splice site by trans-acting factors that undergo quantitative or qualitative changes during differentiation of N2a cells.

Animals↗