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Results for “Alternative Splicing”

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PubMed2001

Modification of alternative splicing of Bcl-x pre-mRNA in prostate and breast cancer cells. analysis of apoptosis and cell death.

There is ample evidence that deregulation of apoptosis results in the development, progression, and/or maintenance of cancer. Since many apoptotic regulatory genes (e.g. bcl-x) code for alternatively spliced protein variants with opposing functions, the manipulation of alternative splicing presents a unique way of regulating the apoptotic response. Here we have targeted oligonucleotides antisense to the 5'-splice site of bcl-x(L), an anti-apoptotic gene that is overexpressed in various cancers, and shifted the splicing pattern of Bcl-x pre-mRNA from Bcl-x(L) to Bcl-x(S), a pro-apoptotic splice variant. This approach induced significant apoptosis in PC-3 prostate cancer cells. In contrast, the same oligonucleotide treatment elicited a much weaker apoptotic response in MCF-7 breast cancer cells. Moreover, although the shift in Bcl-x pre-mRNA splicing inhibited colony formation in both cell lines, this effect was much less pronounced in MCF-7 cells. These differences in responses to oligonucleotide treatment were analyzed in the context of expression of Bcl-x(L), Bcl-x(S), and Bcl-2 proteins. The results indicate that despite the presence of Bcl-x pre-mRNA in a number of cell types, the effects of modification of its splicing by antisense oligonucleotides vary depending on the expression profile of the treated cells.

Alternative Splicing↗

PubMed2026

circASbase: A Comprehensive Database of Alternative Splicing Events in circRNAs.

Although extensive evidence has underscored the critical role of alternative splicing (AS) in generating mature circular RNA (circRNA) isoforms and augmenting their functional diversity, a significant gap remains in the availability of specialized databases housing circRNA AS events. To bridge this gap, we develop circASbase, a pioneering and comprehensive database that catalogs 452,129 AS events in 884,047 full-length circRNAs from 581 samples across 13 species, and provides rich annotations to facilitate understanding the splicing regulation of circRNA. Our findings reveal substantial differences between circRNAs and linear transcripts regarding the distribution and occurrence of AS events, highlighting the unique regulatory landscape of circRNAs. These special splicing events result in functional differences of circRNAs by affecting internal ribosome entry sites, N6-methyladenosine sites, open reading frames, protein features, microRNA targets, and more. In summary, circASbase not only meets the urgent need of the research community for data repositories, but also represents a significant advancement in our understanding of circRNA biology. With its user-friendly interfaces and web-based visualization tools, circASbase is poised to become an indispensable resource for researchers exploring the regulatory mechanisms and functional roles of AS events in circRNAs. This database will continuously drive new insights and discoveries in the field, setting the stage for further advancements in circRNA research. circASbase is freely available at http://reprod.njmu.edu.cn/cgi-bin/circASbase/.

Alternative Splicing↗

PubMed1986

Alternative splicing of SV40 early pre-mRNA in vitro.

Simian virus 40 (SV40) early pre-mRNA is spliced using either of two alternative 5' splice sites and a common 3' splice site to produce two mRNAs that encode the T and t antigens. We have studied alternative splicing of SV40 early pre-mRNA in vitro using a HeLa cell nuclear extract. Synthetic SV40 early transcripts are processed to T and t antigen mRNAs in vitro. As in SV40-infected cells in vivo, cleavage at the T antigen 5' splice site is more efficient than cleavage at the t antigen 5' splice site in vitro, although both of these 5' splice sites are utilized relatively inefficiently in vitro. The ratio of cleavage at the T and t antigen 5' splice sites is not changed significantly by a number of alterations in the conditions under which the in vitro splicing reactions are carried out.

Animals↗

PubMed2005

Placental cathepsin M is alternatively spliced and exclusively expressed in the spongiotrophoblast layer.

Cathepsin M and cathepsin-3 are cysteine peptidases expressed exclusively in the murine placenta. Their expression increases continuously from 11.5 dpc until the end of gestation. The cathepsin M gene consists of 8 exons and 7 introns covering 6 kb of genomic DNA on mouse chromosome 13. Multiple variants of CTSM were identified which display alternative splicing of exon 2 or exon 7. Alternative splicing of exon 2 does not affect the translated region of CTSM whereas aberrant splicing of exon 7 will results in enzymatically inactive versions of CTSM which still might retain inhibitory activity towards cysteine peptidases. Besides two defined major transcription start sites the putative promoter region comprises of a TATA-box and a relatively low (41%) G+C content reflecting its highly specific spatial and temporal expression pattern. Similar features are found within the promoter region of CTS3 which is highly homologous to CTSM. Both cathepsin M and -3 expression are confined to the spongiotrophoblast layer of the mouse placenta an expression pattern which is unique among cysteine peptidases located within the cluster of cathepsin J-like peptidases on mouse chromosome 13.

Alternative Splicing↗

PubMed1997

Tissue specificity and alternative splicing of the Na+/Ca2+ exchanger isoforms NCX1, NCX2, and NCX3 in rat.

The gene coding for the Na+/Ca2+ exchanger NCX1 is characterized by a cluster of six exons (A, B, C, D, E, and F) coding for a variable region in the COOH terminus of the large intracellular loop of the protein. Alternative splicing of these exons generates multiple tissue-specific variants of NCX1. Using reverse transcriptase-polymerase chain reaction, we analyzed eight previously described and four new splicing isoforms of NCX1 in a wide variety of tissues and cells. Exons A and B are mutually exclusive, as shown in earlier studies, and splicing isoforms containing exon A are preferentially expressed in heart, brain, and skeletal muscle, whereas splicing variants with exon B are found in all rat tissues except heart. The second and third isoforms of the Na+/Ca2+ exchanger, NCX2 and NCX3, show a deletion of 37 amino acids in the intracellular loop corresponding to parts of the variable region of NCX1. We identified three splicing isoforms of NCX3 in brain and skeletal muscle by reverse transcriptase-polymerase chain reaction. These splice variants are generated by including either of two alternative exons equivalent to the NCX1 exon A or B and by including or excluding a sequence equivalent to the NCX1 exon C. We did not detect any alternative splicing of NCX2. We examined selected tissues from neonatal and adult rats and found developmental regulation for NCX1 and NCX3 splicing isoforms in skeletal muscle. Specific isoform patterns were also detected for NCX1 and NCX3 in cultured cortical neurons, astrocytes, and oligodendrocytes. We suggest a new terminology to distinguish the different splice variants of individual NCX isoforms.

Aging↗

PubMed1995

Regulation of alternative splicing of protein kinase C beta by insulin.

Insulin regulates a diverse array of cellular signaling processes involved in the control of growth, differentiation, and cellular metabolism. Insulin increases glucose transport via a protein kinase C (PKC)-dependent pathway in BC3H-1 myocytes, but the function of specific PKC isozymes in insulin action has not been elucidated. Two isoforms of PKC beta result via alternative splicing of precursor mRNA. As now shown, both isoforms are present in BC3H-1 myocytes, and insulin induces alternative splicing of the PKC beta mRNA thereby switching expression from PKC beta I to PKC beta II mRNA. This effect occurs rapidly (15 min after insulin treatment) and is dose-dependent. The switch in mRNA is reflected by increases in the protein levels of PKC beta II. High levels of 12-0-tetradecanoylphorbol-13-acetate, which are commonly used to deplete or down-regulate PKC in cells, also induce the switch to PKC beta II mRNA following overnight treatment, and protein levels of PKC beta II reflected mRNA increases. To investigate the functional importance of the shift in PKC beta isoform expression, stable transfectants of NIH-3T3 fibroblasts overexpressing PKC beta I and PKC beta II were established. The overexpression of PKC beta II but not PKC beta I in NIH-3T3 cells significantly enhanced insulin effects on glucose transport. This suggests that PKC beta II may be more selective than PKC beta I for enhancing the glucose transport effects of insulin in at least certain cells and, furthermore, that insulin can regulate the expression of PKC beta II by alternative mRNA splicing.

3T3 Cells↗

PubMed1996

An alternative splicing event in the Pax-3 paired domain identifies the linker region as a key determinant of paired domain DNA-binding activity.

We have identified alternatively spliced isoforms of murine Pax-3 and Pax-7 which differ by the presence or absence of a single glutamine residue in a linker region which separates two distinct DNA-binding subdomains within the paired domain. By reverse transcription-PCR, these isoforms of Pax-3 and Pax-7 (Q+ and Q-) were detected at similar levels through multiple developmental stages in the early mouse embryo. DNA-binding studies using the Q+ and Q- isoforms of Pax-3 revealed that this alternative splicing event had no major effect on the ability of these isoforms to bind to an oligonucleotide specific for the Pax-3 homeodomain (P2) or to a paired domain recognition sequence (e5) that interacts primarily with the N-terminal subdomain of the paired domain. However, DNA-binding studies with sequences (P6CON and CD19-2/A) containing consensus elements for both the N-terminal and C-terminal subdomains revealed that the Q- isoform binds to these sequences with a two- to fivefold-higher affinity; further mutation of the GTCAC core N-terminal subdomain recognition motif of CD19-2/A generated binding sites with a high degree of specificity for the Q- isoform. These differences in DNA binding in vitro were also reflected in the enhanced ability of the Q- isoform to stimulate transcription of a reporter containing multiple copies of CD19-2/A upstream of the thymidine kinase basal promoter. In support of the observations made with these naturally occurring Pax-3 isoforms, introducing a glutamine residue at the analogous position in PAX6 caused a fivefold reduction in binding to P6CON and a complete loss of binding to CD19-2/A and to the C-terminal subdomain-specific probe 5aCON. These studies therefore provide direct evidence for a role for the paired-domain linker region in DNA target site selection, and they identify novel isoforms of Pax-3 and Pax-7 that have the potential to mediate distinct functions in the developing embryo.

Alternative Splicing↗

PubMed1990

Different forms of Ultrabithorax proteins generated by alternative splicing are functionally equivalent.

The Ubx gene of Drosophila normally produces several forms of Ubx proteins through alternative splicing of two microexons. We describe here two new viable Ubx mutations that show similar and almost wild-type adult phenotypes. Molecular characterization has shown that one of them, UbxMX17, is an inversion within the Ubx transcription unit including one of the microexons involved in alternative splicing. This results in mutant flies possessing a very abnormal array of Ubx proteins, probably including spliced forms not present in wild-type flies. Yet these protein products successfully substitute for the normal ones and allow virtually normal Ubx function. We argue that the different Ubx proteins are developmentally equivalent and that the slight mutant phenotype observed in UbxMX17 flies is not due to the abnormal set of Ubx proteins but to a breakpoint in a cis-regulatory region.

Animals↗

PubMed2002

Identification of alternatively spliced Act1 and implications for its roles in oncogenesis.

Act1 (also called CIKS) is a recently identified molecule, which activates NF-kappaB and AP-1. Here, we identified alternatively spliced Act1 that lacked the exon 2 encoding the first nine amino acids in the amino terminus of the protein. Compared to full-length Act1, this truncated Act1 appeared to be equally active. We demonstrated further that only the spliced Act1 was detected in cDNA libraries derived from human fetal brain, liver, leukocytes, and bone marrow. In contrast, both the spliced and full-length Act1 templates were detected in a variety of human cancer cell lines. The expression of both the spliced and full-length transcripts was detected at 4-h time point, following the treatment of endothelial cells with tumor necrosis factor-alpha, interleukin-1beta or bacterial endotoxin. Notably, the dominant amounts of the spliced Act1 over the full-length Act1 were amplified from both the cancer cell mRNAs and the stimulated endothelial cell mRNAs. Taken together with the act1 chromosome localization at the 6q21 subregion, our findings indicate that the newly identified alternatively spliced Act1 is a major transcript of the molecule and that Act1 may play important roles in oncogenesis.

Adaptor Proteins, Signal Transducing↗

PubMed1995

Developmental role of transcription factor isoforms generated by alternative splicing.

The production of transcription factor isoforms by developmentally regulated alternative splicing of pre-mRNAs is a widespread phenomenon. Frequently, differences in biochemical function among the isoforms can be predicted from sequence analysis, and in many instances such differences have been demonstrated in vitro or in cultured cells. A great variety of strategies for functional diversification can be classified into three main types: modulation of DNA binding specificity or affinity, production of activators and antagonists from the same gene, and modulation of dimerization properties. Despite obvious implications in many cases, the actual developmental consequences are understood only in a few instances. The roles inferred from these examples are diverse, ranging from developmental switches that have profound effects on pathways of differentiation to mechanisms that may optimize or fine-tune transcription factor function in different contexts.

Alternative Splicing↗

PubMed2004

Oligomer-mediated modulation of hTERT alternative splicing induces telomerase inhibition and cell growth decline in human prostate cancer cells.

The expression of telomerase in human cells is strictly controlled by multiple mechanisms including transcription and alternative splicing of telomerase reverse transcriptase (hTERT). In this study, we demonstrated the possibility of modulating the hTERT splicing pattern in DU145 human prostate carcinoma cells through the use of 2'-O-methyl-RNA phosphorothioate oligonucleotides targeting the splicing site located between intron 5 and exon 6 in the hTERT pre-mRNA. An 18-h oligonucleotide exposure induced a decrease in the full-length hTERT transcript and a concomitant increase in the alternatively spliced transcripts, which resulted in significant inhibition of telomerase catalytic activity. Moreover, exposure to the R7 oligomer (which induced the most pronounced modulation of the hTERT splicing pattern and the greatest telomerase inhibition) caused a marked reduction in DU145 cell growth and the induction of apoptosis starting 2 days after treatment. Such data support the concept that down-regulation of hTERT expression can cause short-term effects on tumour cell growth, which are telomere-shortening independent.

Alternative Splicing↗

PubMed2026

Alternative splicing of toll-like receptor pathway mRNAs in lung immune cells from patients with ARDS.

Acute respiratory distress syndrome (ARDS) is characterized by robust inflammation in the lungs and systemic circulation. In this context, the toll-like receptor (TLR) signaling pathway plays a major role, driving inflammation that promotes host defense but also causing pathological tissue damage. To limit excessive inflammation, TLR signaling must be tightly controlled. One mechanism that modulates TLR signaling is alternative splicing of TLR pathway pre-mRNAs, which balances production of positively acting inflammatory mediators with alternative splice forms that terminate inflammation. To determine whether altered TLR pathway splicing contributes to pathological inflammation in ARDS, we evaluated two central mediators of the TLR signaling pathway, the MyD88 signaling adapter and the IRAK1 signaling kinase, in leukocytes isolated from bronchoalveolar lavage (BAL) of patients with ARDS. We found that MyD88 gene expression was decreased in BAL immune cells, whereas IRAK1 gene expression was increased. In parallel, we monitored long proinflammatory (MyD88-L and IRAK1) and shorter anti-inflammatory (MyD88-S and IRAK1c) splice forms and determined that IRAK1 splicing was shifted in a proinflammatory direction in patients with ARDS. Finally, we evaluated relationships between MyD88 isoform levels in BAL leukocytes and clinical outcomes. We conclude that pre-mRNA splicing of TLR pathway genes is altered in lung immune cells in patients with ARDS, that monitoring splicing of these genes may provide important prognostic information, and that manipulating splicing of these genes may be a useful novel therapeutic approach that needs further investigation.NEW & NOTEWORTHY We found that MyD88 expression is decreased, that IRAK1 expression is increased, and that IRAK1 splicing is shifted in a proinflammatory direction, in lung immune cells in patients with ARDS. We also find that MyD88 expression levels may correlate with survival in patients with ARDS. Thus, changes in expression and splicing of these two genes offer potential novel prognostic and therapeutic targets for ARDS.

Humans↗

PubMed1997

Alternative splice variants of cytokines: making a list.

The cytokine network is an extremely complicated and redundant system of mutually interdependent pleiotropic cytokines that interacts with a variety of cells, tissues and organs producing various regulatory effects, both local and systemic. Recent studies have revealed a new source of complexity in the cytokine network--alternative splicing. This minireview attempts to summarize the available data on the alternative splicing of cytokines and its regulatory significance.

Alternative Splicing↗

PubMed1997

Plasma membrane Ca2+ pump in rat brain. Patterns of alternative splices seen by isoform-specific antibodies.

The expression at the protein level of plasma membrane calcium pump (PMCA) isoforms in rat brain was detected by new antibodies that distinguished the four gene products and their alternatively spliced variants. All four gene products were distributed throughout hippocampus, cortex, and cerebellum, but the alternate splices showed more distinct distribution patterns. The b splice of isoform 1 was not detectable in any of the brain regions, which makes it unlikely that this isoform performs an essential housekeeping role as is frequently proposed. The b splices of isoforms 3 and 4, although expressed in all three regions, showed evidence of proteolysis, which removed a portion of the carboxyl terminus. In contrast, isoform 2b retained its full length, indicating that PMCA2b is more resistant to proteolysis than the other b forms. Whereas substantial amounts of isoforms 1a, 2a, and 3a were expressed in all regions, 4a was found only in frontal cortex. The distinct patterns of expression of the PMCA isoforms in brain suggest that some of them play a special role in intracellular Ca regulation.

Alternative Splicing↗

PubMed1992

Polypyrimidine tract binding protein interacts with sequences involved in alternative splicing of beta-tropomyosin pre-mRNA.

Previous studies of alternative splicing of the rat beta-tropomyosin gene have shown that nonmuscle cells contain factors that block the use of the skeletal muscle exon 7 (Guo, W., Mulligan, G. J., Wormsley, S., and Helfman, D. M. (1991) Genes & Dev. 5, 2095-2106). Using an RNA mobility-shift assay we have identified factors in HeLa cell nuclear extracts that specifically interact with sequences responsible for exon blockage. Here we present the purification to apparent homogeneity of a protein that exhibits these sequence specific RNA binding properties. This protein is identical to the polypyrimidine tract binding protein (PTB) which other studies have suggested is involved in the recognition and efficient use of 3'-splice sites. PTB binds to two distinct functional elements within intron 6 of the beta-tropomyosin pre-mRNA: 1) the polypyrimidine tract sequences required for the use of branch points associated with the splicing of exon 7, and 2) the intron regulatory element that is involved in the repression of exon 7. Our results demonstrate that the sequence requirements for PTB binding are different than previously reported and shows that PTB binding cannot be predicted solely on the basis of pyrimidine content. In addition, PTB fails to bind stably to sequences within intron 5 and intron 7 of beta-TM pre-mRNA, yet forms a stable complex with sequences in intron 6, which is not normally spliced in HeLa cells in vitro and in vivo. The nature of the interactions of PTB within this regulated intron reveals several new details about the binding specificity of PTB and suggests that PTB does not function exclusively in a positive manner in the recognition and use of 3'-splice sites.

Alternative Splicing↗

PubMed1997

DNA binding specificity of proteins derived from alternatively spliced mouse p53 mRNAs.

The mouse p53 gene generates two alternative splice products encoding p53 protein and a naturally occurring protein (p53as) with changes at the C-terminus. In p53as the negative regulatory region for DNA binding and PAb421 antibody binding site are replaced, and p53as is constitutively active for sequence-specific DNA binding. Using the technique of randomized synthetic oligonucleotide in cyclic amplification and selection of targets, we have found that p53as and p53 proteins have the same DNA binding specificities but that these specificities frequently diverge from the consensus of two copies of PuPuPuCATGPyPyPy. The importance of tetranucleotide CATG was confirmed but there was a less rigorous requirement for patterns of flanking or intervening sequences. In particular, the three purines upstream and three pyrimidines downstream of CATG are not required for p53 or p53as binding, 29 or more intervening nucleotides are tolerated, and one CATG is sufficient where adjacent nucleotides contain a region of homology with certain previously reported non-consensus p53 binding sequences. These results suggested further definition of the non-consensus motifs, and database searches with these uncovered additional candidate genes for p53 protein binding. We conclude that p53as and perhaps other activated forms of p53 exert their effects on the same genes and that differential activities of p53 protein forms are not due to inherently different sequence selectivities of DNA binding.

Alternative Splicing↗

PubMed2001

Differential alternative splicing activity of isoforms of polypyrimidine tract binding protein (PTB).

Polypyrimidine tract binding protein (PTB) is an RNA-binding protein that regulates splicing by repressing specific splicing events. It also has roles in 3'-end processing, internal initiation of translation, and RNA localization. PTB exists in three alternatively spliced isoforms, PTB1, PTB2, and PTB4, which differ by the insertion of 19 or 26 amino acids, respectively, between the second and third RNA recognition motif domains. Here we show that the PTB isoforms have distinct activities upon alpha-tropomyosin (TM) alternative splicing. PTB1 reduced the repression of TM exon 3 in transfected smooth muscle cells, whereas PTB4 enhanced TM exon 3 skipping in vivo and in vitro. PTB2 had an intermediate effect. The PTB4 > PTB2 > PTB1 repressive hierarchy was observed in all in vivo and in vitro assays with TM, but the isoforms were equally active in inducing skipping of alpha-actinin exons and showed the opposite hierarchy of activity when tested for activation of IRES-driven translation. These findings establish that the ratio of PTB isoforms could form part of a cellular code that in turn controls the splicing of various other pre-mRNAs.

Alternative Splicing↗

PubMed2002

Functional characterization of alternatively spliced 5-HT2 receptor isoforms from the pharynx and muscle of the parasitic nematode, Ascaris suum.

Serotonin (5-HT) receptors play key regulatory roles in nematodes and alternatively spliced 5-HT2 receptor isoforms have been identified in the parasitic nematode, Ascaris suum. 5-HT2As1 and 5-HT2As2 contain different C-termini, and 5-HT2As1Delta4 lacks 42 amino acids at the C-terminus of the third intracellular loop. 5-HT2As1 and 5-HT2As2 exhibited identical pharmacological profiles when stably expressed in human embryonic kidney (HEK) 293 cells. Both 5-HT2As isoforms had higher affinity for 5-HT than their closely related Caenorhabditis elegans homolog (5-HT2Ce). This increased 5-HT affinity was not related to the substitution in 5-HT2As1 of F120 for Y in the highly conserved DRY motif found in the second intracellular loop of other 5-HT receptors, since a 5-HT2As1F120Y mutant actually exhibited increased 5-HT affinity compared with that of 5-HT2As1. As predicted, cells expressing either 5-HT2As1 or 5-HT2As2 exhibited a 5-HT-dependent increase in phosphatidylinositol (PI) turnover. In contrast, although 5-HT2As1Delta4 displayed a 10-fold higher affinity for 5-HT and 5-HT agonists than either 5-HT2As1 or 5-HT2As2, 5-HT2As1Delta4 did not couple to either PI turnover or adenyl cyclase activity. Based on RT-PCR, 5-HT2As1 and 5-HT2As2 were more highly expressed in pharynx and body wall muscle and 5-HT2As1Delta4 in nerve cord/hypodermis. This is the first report of different alternatively spliced 5-HT2 receptor isoforms from any system.

Alternative Splicing↗