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PubMed1996

Adhesion mediated by fibronectin's alternatively spliced EDb (EIIIB) and its neighboring type III repeats.

Adhesion functions of cellular fibronectin's (FN) alternatively spliced EDb (EIIIB) domain, as well as its neighboring type III repeats III7 and III8, were investigated with several cultured murine and human cell types. Minigene constructs encoding various permutations of these repeats and expressed in bacteria were used as shown previously in function studies of EDa and its neighboring repeats (P.Xia and L. A. Culp Exp. Cell Res. 213, 253-265, 1994). When substrata of recombinant proteins were incubated with several fibroblastic or neuronal derivative cell lines, cell attachment responses varied widely in a cell-type-specific manner. Balb/c 3T3 cells were shown to adhere to recombinant protein substrata in dose-dependent and EDTA-sensitive manners. Responses also varied with which repeat combinations were being tested, from excellent (Balb/c 3T3, src-3T3), to intermediate (Platt cells), to poor (LZEJ, VA-13, and F11), with EDb plus-containing proteins generally giving better adhesion than EDb minus proteins. On select recombinant proteins, cells showed limited cytoplasmic spreading (3T3 and src-3T3) or neurite extension (Platt and F11), while other cell lines (VA-13 and LZEJ) did not show any morphological changes beyond attachment. Again, EDb plus-containing recombinants were more effective at inducing these morphological changes than the neighboring repeats. These results demonstrate that the EDb domain of cellular FNs and its neighboring type III homology repeats contain important adhesion-promoting sequences, which may be regulated by cells through alternative splicing of FN's primary transcript.

Alternative Splicing↗

PubMed2002

Categorization and characterization of transcript-confirmed constitutively and alternatively spliced introns and exons from human.

By spliced alignment of human DNA and transcript sequence data we constructed a data set of transcript-confirmed exons and introns from 2793 genes, 796 of which (28%) were seen to have multiple isoforms. We find that over one-third of human exons can translate in more than one frame, and that this is highly correlated with G+C content. Introns containing adenosine at donor site position +3 (A3), rather than guanosine (G3), are more common in low G+C regions, while the converse is true in high G+C regions. These two classes of introns are shown to have distinct lengths, consensus sequences and correlations among splice signals, leading to the hypothesis that A3 donor sites are associated with exon definition, and G3 donor sites with intron definition. Minor classes of introns, including GC-AG, U12-type GT-AG, weak, and putative AG-dependant introns are identified and characterized. Cassette exons are more prevalent in low G+C regions, while exon isoforms are more prevalent in high G+C regions. Cassette exon events outnumber other alternative events, while exon isoform events involve truncation twice as often as extension, and occur at acceptor sites twice as often as at donor sites. Alternative splicing is usually associated with weak splice signals, and in a majority of cases, preserves the coding frame. The reported characteristics of constitutive and alternative splice signals, and the hypotheses offered regarding alternative splicing and genome organization, have important implications for experimental research into RNA processing. The 'AltExtron' data sets are available at http://www.bit.uq.edu.au/altExtron/ and http://www.ebi.ac.uk/~thanaraj/altExtron/.

AT Rich Sequence↗

PubMed1996

Expression of CD44 and the pattern of CD44 alternative splicing in uveal melanoma.

In cutaneous melanoma, the standard CD44 molecule is abundantly expressed, whereas the expression of certain splice variants is related to tumour progression and to the metastatic potential of the cell line. In the present study we have investigated the expression of CD44 and the pattern of CD44 alternative splicing in uveal melanoma in relation to the cell type, diameter and invasiveness of the tumour. All uveal melanomas strongly stained with antibodies to the standard portion of CD44. No expression of the CD44 variant (v) exon CD44v7 was found, whereas v5, v6 and v10 were expressed (in 2/12, 5/12 and 8/12 cases, respectively). No correlation was observed between expression of particular splice variants and cell type, tumour diameter or invasion of the sclera or Bruch's membrane. All three uveal melanoma cell lines tested were strongly CD44 positive and expressed low levels of v6-containing isoforms at the cell surface, but v5, v7 and v10 were absent. Our results show that CD44 is strongly expressed in uveal melanoma and that the pattern of CD44 alternative splicing is similar to that observed in cutaneous melanoma. However, in uveal melanoma this alternative splicing does not appear to be related to prognostic parameters.

Alternative Splicing↗

PubMed1989

Sex-specific alternative splicing of RNA from the transformer gene results from sequence-dependent splice site blockage.

Sex-specific alternative splicing of RNA from the Drosophila transformer gene involves competition between two 3' splice sites. In the absence of Sex-lethal activity (as in males), only one site functions; in the presence of Sex-lethal activity (as in females), both sites function. Information for sex-specific splice site choice is contained within the intron itself. Deletions of the splice site used in males lead to Sex-lethal-independent use of the otherwise female-specific site. The relative amounts of unspliced and spliced RNA derived from these mutant genes do not change with changes in Sex-lethal activity. Specific nucleotide changes in the non-sex-specific splice site do not affect splicing activity but eliminate Sex-lethal-induced regulation. A deletion removing material between the two splice sites does not eliminate sex-specific regulation, while a deletion of the female splice site leads to a female-specific increase in unspliced RNA. These results are consistent with a model in which female-specific factors block the function of the non-sex-specific 3' splice site.

Animals↗

PubMed2004

Activation of c-myb by 5' retrovirus promoter insertion in myeloid neoplasms is dependent upon an intact alternative splice donor site (SD') in gag.

Alternative splicing in Mo-MuLV recruits a splice donor site, SD', within the gag that is required for optimal replication in vitro. Remarkably, this SD' site was also found to be utilized for production of oncogenic gag-myb fusion RNA in 100% of murine-induced myeloid leukemia (MML) in pristane-treated BALB/c mice. Therefore, we investigated the influence of silent mutations of SD' in this model. Although there was no decrease in the overall incidence of disease, there was a decrease in the incidence of myeloid leukemia with a concomitant increase in lymphoid leukemia. Importantly, there was a complete lack of myeloid tumors associated with 5' insertional mutagenic activation of c-myb, suggesting the specific requirement of the SD' site in this mechanism.

Animals↗

PubMed1995

Alternative splicing at the C-terminal but not at the N-terminal domain of the NMDA receptor NR1 is altered in the kindled hippocampus.

Several lines of evidence suggest that N-methyl-D-aspartate (NMDA) receptors significantly contribute to the development of kindling. In addition, a lasting enhancement of the NMDA receptor function has been suggested to play a significant role in the chronic hyperexcitability occurring in the hippocampus after kindling epileptogenesis. We have investigated whether hippocampal kindling induces changes in the NMDA receptor at the molecular level by assessing the expression of mRNAs of the different spliced variants at the N-terminal (exon 5) and C-terminal (exon 21) position of the NMDA receptor 1 (NR1) gene by means of the reverse transcription-polymerase chain reaction. Alternative splicing at exon 5 confers different sensitivity of the NMDA receptor to polyamines while exon 21 encodes a 37-amino acid insert containing the major phosphorylation sites for protein kinase C. One week after the acquisition of stage 5 of kindling in rats (generalized tonic-clonic seizures), the relative abundance of the two alternatively spliced forms at the C-terminal domain, respectively containing (+) or lacking (-) exon 21, was reversed compared to controls (implanted with electrodes but not stimulated) in the dorsal hippocampus ipsilateral and contralateral to the electrical stimulation. The exon 21+/exon 21- mRNA ratio for controls was 1.3 +/- 0.04 (mean +/- SE); for ipsilaterally kindled rats it was 0.64 +/- 0.05 (P < 0.05), and for contralaterally kindled rats it was 0.48 +/- 0.07 (P < 0.01). Similar bilateral effects were observed in the ventral hippocampus (temporal pole). No changes were found 4 weeks after stage 5 seizures and 1 week after the induction of a single afterdischarge. No significant alterations were induced by kindling in the relative abundance of the spliced variants containing or lacking exon 5. Our findings show selective changes in alternative splicing of the NR1 gene after repeated application of an epileptogenic stimulus. This may generate receptors with different functional properties, which may contribute to the increased sensitivity for the induction of generalized seizures during kindling.

Alternative Splicing↗

PubMed1995

Alternative splicing of calretinin mRNA leads to different forms of calretinin.

cDNA clones for calretinin, a member of the troponin-C family of calcium-binding proteins, were isolated from a cDNA library of the human colon carcinoma cell line WiDr. Sequence analysis revealed two forms of alternatively spliced calretinin mRNAs encoding C-terminally truncated proteins. Exon 7 was either spliced to exon 9 (delta 8) or to exon 10 (delta 8,9); both resulted in a frame shift and a translational stop at the second codon of exon 9 (delta 8), or at codon 15 of exon 10 (delta 8,9), respectively. The presence of delta 8 and delta 8,9 calretinin mRNA in WiDr cells was confirmed using reverse-transcriptase PCR and sequence analysis of the amplicon, as well as by a ribonuclease protection assay. Co115/3 and three other human colon carcinoma cell lines were found, by reverse-transcriptase PCR to also contain delta 8,9 calretinin mRNA. The truncated proteins were able to bind calcium, as evidenced by a calcium blot of the delta 8 form (calretinin-20k) and delta 8,9 form (calretinin-22k) expressed in Escherichia coli. Immunohistochemical staining using an antiserum specific for the novel C-terminus of calretinin-22k confirmed its presence in WiDr, Co115/3 and three additional colon carcinoma cell lines. The fact that alternative splicing of calretinin was found in five different cell lines suggests that alternatively spliced calretinins fulfill a physiological function.

Alternative Splicing↗

PubMed1993

In vitro analysis of bovine growth hormone pre-mRNA alternative splicing. Involvement of exon sequences and trans-acting factor(s).

Bovine growth hormone (bGH) pre-mRNA is alternatively spliced, resulting in retention of the last intron (intron D) in a fraction of the cytosolic bGH mRNA. To study the mechanism of this alternative splicing event, we examined the splicing of bGH pre-mRNA in vitro. The splicing of bGH intron D in vitro required a 115-base pair segment of exon 5, reflecting the positive influence of exon sequences observed in transfected cells. No detectable spliceosome complex formation was observed using bGH pre-mRNA containing the 115-base pair deletion in exon 5. The in vitro splicing of the wild type bGH pre-mRNA was inhibited by the addition of RNA containing the 115-nucleotide exon sequence, but not by nonspecific RNAs. UV irradiation of the in vitro splicing reaction resulted in specific cross-linking of a 35-kDa protein(s) to the 115-nucleotide bGH exon sequence. These results suggest that terminal exon sequences are required at an early step of spliceosome complex formation and are consistent with a mechanism in which saturable, trans-acting factor(s) bind to these exon sequences to activate spliceosome complex formation and splicing of bGH intron D.

Alternative Splicing↗

PubMed2003

Alternatively spliced products of the human kinesin light chain 1 (KNS2) gene.

Conventional kinesin is a microtubule-based molecular motor involved in the transport of membranous and non-membranous cargoes. The kinesin holoenzyme exists as a heterotetramer, consisting of two heavy chain and two light chain subunits. It is thought that one function of the light chains is to interact with the cargo. Alternative splicing of kinesin light chain pre-mRNA has been observed in lower organisms, although evidence for alternative splicing of the human gene has not been reported. We have identified 19 variants of the human KNS2 gene (KLC1) that are generated by alternative splicing of downstream exons, but calculate that KNS2 has the potential to produce 285 919 spliceforms. Corresponding spliceforms of the mouse KLC1 gene were also identified. The alternative exons are all located 3' of exon 12 and the novel spliceforms produce both alternative carboxy termini and alternative 3' untranslated regions. The observation of multiple light chain isoforms is consistent with their proposed role in specific cargo attachment.

Alternative Splicing↗

PubMed1993

Location and alternative splicing of type XIII collagen RNA in the early human placenta.

BACKGROUND: Type XIII collagen is widely distributed in the fetus. It is characterized by complex alternative splicing of its primary transcripts in regions corresponding to nine exons of the gene. EXPERIMENTAL DESIGN: Localization of type XIII collagen mRNAs in early placentas was determined by in situ hybridization. Reverse-transcription-polymerase chain reactions were used to examine alternative splicing of nine exons in villous and decidual mRNAs. RESULTS: An intense in situ hybridization signal was observed in the fibroblastoid stromal cells of the placental villi. A moderate signal was found in the endothelial cells of developing capillaries and the cells of the cytotrophoblastic columns. Furthermore, mRNAs were detected in the large decidual cells of the decidual membrane and the stromal cells of the gestational endometrium, but not in the epithelial cells in the endometrial glands. Five combinations of exons 3B, 4A, 4B and 5, encoding half of the COL1 domain, were found. The combination lacking exons 3B-5 was the major variant in both villous and decidual mRNAs. Three combinations of exons 12 and 13 encoding the NC2 domain were found, the long variant containing either 12 or 13 sequences being the major variant in the villi while nearly equal amounts of long and short variants lacking both 12 and 13 sequences were observed in the decidua. Four variants of exons 29, 33 and 37, encoding parts of COL3 and NC4, were found as splicing out of exon 37 was not detected. The major variants in both mRNAs were two that lacked exon 29 and either lacked or contained exon 33 sequences. CONCLUSIONS: Type XIII collagen mRNAs were located in the placenta. Due to alternative splicing, the lengths of the COL1 and NC2 domains of this collagen vary from 57 to 104 and from 12 to 34 amino acids, respectively. The COL3 domain varies between 208 and 235 residues, whereas the NC4 is 18 residues.

Base Sequence↗

PubMed2004

Branch site haplotypes that control alternative splicing.

We show that the allele-dependent expression of transcripts encoding soluble HLA-DQbeta chains is determined by branchpoint sequence (BPS) haplotypes in DQB1 intron 3. BPS RNAs associated with low inclusion of the transmembrane exon in mature transcripts showed impaired binding to splicing factor 1 (SF1), indicating that alternative splicing of DQB1 is controlled by differential BPS recognition early during spliceosome assembly. We also demonstrate that naturally occurring human BPS point mutations that alter splicing and lead to recognizable phenotypes cluster in BP and in position -2 relative to BP, implicating impaired SF1-BPS interactions in disease-associated BPS substitutions. Coding DNA variants produced smaller fluctuations of exon inclusion levels than random exonic substitutions, consistent with a selection against coding mutations that alter their own exonization. Finally, proximal splicing in this multi-allelic reporter system was promoted by at least seven SR proteins and repressed by hnRNPs F, H and I, supporting an extensive antagonism of factors balancing the splice site selection. These results provide the molecular basis for the haplotype-specific expression of soluble DQbeta, improve prediction of intronic point mutations and indicate how extraordinary, selection-driven DNA variability in HLA affects pre-mRNA splicing.

Alternative Splicing↗

PubMed1998

The role of alternative splicing in regulating agrin binding to muscle cells.

The binding of agrin to the muscle cell surface can induce radical changes in the topography and physiology of the cell membrane, resulting in the organization of postsynaptic components opposite the nerve terminal. Alternative splicing of agrin mRNA yields several isoforms, which vary in their cellular expression, developmental profile, and acetylcholine receptor (AChR) clustering activity. Neurons and muscle cells express several of these agrin isoforms. To address the role of alternative splicing in regulating agrin's function, we compared the effects of splicing at the y and z sites of agrin (denoted 'Agy,z'). Agrin isoforms bound differently to the myotube surface: Ag0,0 and Ag4,0 showed much higher levels of binding than Ag4,8. The artificial splice form Ag0,8 showed binding levels similar to Ag4,8. Visualization of the bound agrin after an acute incubation revealed that each isoform associated with the cell surface in a distinct pattern. These binding patterns changed following stimulation of the myotubes with Ag4,8 for 4 h (which induces the clustering of AChRs). Ag4,8 binding sites were concentrated at >90% of the induced AChR clusters, while those for Ag4,0, Ag0,8, and Ag0,0 were enriched at 70%, 50% and 25%, respectively. Together, these observations indicate that alternatively spliced forms of agrin recognize at least partially non-overlapping populations of binding sites on the cell surface, and that the eight amino acid insert is the dominant factor influencing the level of the agrin binding to the cell surface. Further, some of these populations redistribute to AChR clusters upon agrin stimulation.

Agrin↗

PubMed1994

cDNA cloning of the rat IGF I receptors: structural analysis of rat and human IGF I and insulin receptors reveals differences in alternative splicing and receptor-specific domain conservation.

IGF I and insulin receptors are homologous proteins that function in distinct physiological pathways. To define domains that might contribute to differences between IGF I and insulin receptors, we cloned the rat IGF I receptor cDNA and performed a comparative sequence analysis of specific functional domains in the two receptor types of rats and humans. Since alternative splicing has been shown to alter the activities of both IGF I and insulin receptors, we also examined the mRNA splicing patterns of the two receptors. The C-terminal region exhibits the lowest degree of amino acid homology between rat and human IGF I receptors (85%) and the tyrosine kinase domain the highest homology (98%). In the region corresponding to the CAG+/-alternative splicing site of the human IGF I receptor, a nucleotide change in the rat eliminates the alternative acceptor splice site. The rat IGF I receptor has no equivalent to the alternatively spliced exon 11 of the insulin receptor. The IGF I and insulin receptors are highly homologous in the tyrosine kinase domain (84%), but differ markedly in other specific regions (e.g., 22-26% homology in the transmembrane domain, 45% homology in the C-terminal domain). We speculate that these regions of divergent sequence may have roles in determining distinct signaling properties of IGF I and insulin receptors.

Alternative Splicing↗

PubMed1997

Organization of the human protein 4.1 genomic locus: new insights into the tissue-specific alternative splicing of the pre-mRNA.

Protein 4.1 is a globular 80-kDa component of the erythrocyte membrane skeleton that enhances spectrin-actin interaction via its internal 10-kDa domain. Previous studies have shown that protein 4.1 mRNA is expressed as multiple alternatively spliced isoforms, resulting from the inclusion or exclusion of small cassette sequences called motifs. By tissue screening for protein 4.1 isoforms, we have observed new features of an already complex pattern of alternative splicing within the spectrin/actin binding domain. In particular, we found a new 51-nt exon that is present almost exclusively in muscle tissue. In addition, we have isolated multiple genomic clones spanning over 200 kb, containing the entire erythroid and nonerythroid coding sequence of the human locus. The exon/intron structure has now been characterized; with the exception of a 17-nt motif, all of the alternatively spliced motifs correspond to individual exons. The 3'-untranslated region (UTR) has also been completely sequenced using various PCR and genomic-sequencing methods. The 3' UTR, over 3 kb, accounts for one-half of the mature mRNA.

Actins↗

PubMed1998

Structure and alternative splicing of the ketohexokinase gene.

Ketohexokinase (fructokinase, KHK) catalyses the phosphorylation of fructose to fructose-l-phosphate. It thereby initiates the intracellular catabolism of a large proportion of dietary carbohydrate. Although found at high level in liver, renal cortex and small intestine, fructokinase activity has also been known for many years to be present at lower levels in most other tissues. We previously found that there appeared to be two isoforms of human KHK, and have now investigated the molecular basis for this in human, rat and mouse. Cloning of the human KHK gene, on chromosome 2p23.2-2p23.3, shows that it has nine exons, spanning 14 kb. An intragenic duplication has resulted in two similar 135-bp exons (designated 3a and 3c), separated by a short intron. Exon 3a and exon 3c are mutually exclusively spliced into KHK mRNA. This exon-intron structure and the pattern of alternative splicing are conserved in both the rat and mouse, suggesting distinct conserved functions for the two KHK isoforms. The alternative splicing is also tissue specific, since in both rat and human, tissues expressing high levels of KHK (liver, kidney and duodenum) utilise exclusively the 3c exon, while other tissues use only 3a. Furthermore, comparison of human foetal and adult tissues indicates a developmental splicing shift from use of exon 3a to exon 3c.

Alternative Splicing↗

PubMed2000

C-Terminal alternative splicing changes the gating properties of a human spinal cord calcium channel alpha 1A subunit.

The calcium channel alpha(1A) subunit gene codes for proteins with diverse structure and function. This diversity may be important for fine tuning neurotransmitter release at central and peripheral synapses. The alpha(1A) C terminus, which serves a critical role in processing information from intracellular signaling molecules, is capable of undergoing extensive alternative splicing. The purpose of this study was to determine the extent to which C-terminal alternative splicing affects some of the fundamental biophysical properties of alpha(1A) subunits. Specifically, the biophysical properties of two alternatively spliced alpha(1A) subunits were compared. One variant was identical to an isoform identified previously in human brain, and the other was a novel isoform isolated from human spinal cord. The variants differed by two amino acids (NP) in the extracellular linker between transmembrane segments IVS3 and IVS4 and in two C-terminal regions encoded by exons 37 and 44. Expression in Xenopus oocytes demonstrated that the two variants were similar with respect to current-voltage relationships and the voltage dependence of steady-state activation and inactivation. However, the rates of activation, inactivation, deactivation, and recovery from inactivation were all significantly slower for the spinal cord variant. A chimeric strategy demonstrated that the inclusion of the sequence encoded by exon 44 specifically affects the rate of inactivation. These findings demonstrate that C-terminal structural changes alone can influence the way in which alpha(1A) subunits respond to a depolarizing stimulus and add to the developing picture of the C terminus as a critical domain in the regulation of Ca(2+) channel function.

Alternative Splicing↗

PubMed2020

Exploration of predictive and prognostic alternative splicing signatures in lung adenocarcinoma using machine learning methods.

BACKGROUND: Alternative splicing (AS) plays critical roles in generating protein diversity and complexity. Dysregulation of AS underlies the initiation and progression of tumors. Machine learning approaches have emerged as efficient tools to identify promising biomarkers. It is meaningful to explore pivotal AS events (ASEs) to deepen understanding and improve prognostic assessments of lung adenocarcinoma (LUAD) via machine learning algorithms. METHOD: RNA sequencing data and AS data were extracted from The Cancer Genome Atlas (TCGA) database and TCGA SpliceSeq database. Using several machine learning methods, we identified 24 pairs of LUAD-related ASEs implicated in splicing switches and a random forest-based classifiers for identifying lymph node metastasis (LNM) consisting of 12 ASEs. Furthermore, we identified key prognosis-related ASEs and established a 16-ASE-based prognostic model to predict overall survival for LUAD patients using Cox regression model, random survival forest analysis, and forward selection model. Bioinformatics analyses were also applied to identify underlying mechanisms and associated upstream splicing factors (SFs). RESULTS: Each pair of ASEs was spliced from the same parent gene, and exhibited perfect inverse intrapair correlation (correlation coefficient = - 1). The 12-ASE-based classifier showed robust ability to evaluate LNM status of LUAD patients with the area under the receiver operating characteristic (ROC) curve (AUC) more than 0.7 in fivefold cross-validation. The prognostic model performed well at 1, 3, 5, and 10 years in both the training cohort and internal test cohort. Univariate and multivariate Cox regression indicated the prognostic model could be used as an independent prognostic factor for patients with LUAD. Further analysis revealed correlations between the prognostic model and American Joint Committee on Cancer stage, T stage, N stage, and living status. The splicing network constructed of survival-related SFs and ASEs depicts regulatory relationships between them. CONCLUSION: In summary, our study provides insight into LUAD researches and managements based on these AS biomarkers.

Adenocarcinoma of Lung↗

PubMed1997

Dp140: alternatively spliced isoforms in brain and kidney.

Dp140, a protein composed of the distal rod domain and carboxy-terminal domain of dystrophin, is expressed only in brain and kidney; transcription is initiated at a unique first exon located in dystrophin intron 44. Both tissues express specific isoforms with distinct alternative splicing of exons 71-74 and 78. The carboxy-terminal domain of Dp140 is identical to that of full-length 427-kDa dystrophin, and it is this region that is associated, with the "dystrophin-associated protein" complex in skeletal muscle. Alternative splicing encodes domains that have been shown to be protein-binding regions; thus this alternative splicing may regulate the association of Dp140 with integral membrane proteins. The 5' flanking region of genomic DNA adjacent to the Dp140 first exon contains a variety of transcription factor-binding motifs, some of which could regulate neuroglial-specific gene expression.

Alternative Splicing↗