Alpha-tropomyosin mutually exclusive exon selection: competition between branchpoint/polypyrimidine tracts determines default exon choice.

Mullen, Michael P.; Smith, Christopher W.; Patton, James G.; Nadal-Ginard, B

doi:10.1101/gad.5.4.642

Cited by 148 publications

(136 citation statements)

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“…The selection of exon 2a is therefore considered to be an SMC-specific event. The splicing mechanism of the ␣-TM genes has been also studied (37,38); however, there are no reports regarding isoform interconversion of TM in association with phenotypic modulation of SMCs.…”

mentioning

confidence: 99%

Coordinate Expression of α-Tropomyosin and Caldesmon Isoforms in Association with Phenotypic Modulation of Smooth Muscle Cells

Kashiwada¹,

Nishida²,

Hayashi³

et al. 1997

Journal of Biological Chemistry

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Isoform diversity of tropomyosin is generated from the limited genes by a combination of differential transcription and alternative splicing. In the case of the ␣-tropomyosin (␣-TM) gene, exon 2a rather than exon 2b is specifically spliced in ␣-TM-SM mRNA, which is one of the major tropomyosin isoforms in smooth muscle cells. Here we demonstrate that expressions of ␣-tropomyosin and caldesmon isoforms are coordinately regulated in association with phenotypic modulation of smooth muscle cells. Molecular cloning and Western and Northern blottings have revealed that in addition to the downregulation of ␤-TM-SM, ␣-TM-SM converted to ␣-TM-F1 and ␣-TM-F2 by a selectional change from exon 2a to exon 2b during dedifferentiation of smooth muscle cells in culture. Simultaneously, a change of caldesmon isoforms from high M r type to low M r type was also observed by alternative selection between exons 3b and 4 in the caldesmon gene during this process. In contrast, cultured smooth muscle cells maintaining a differentiated phenotype continued to express ␣-TM-SM, ␤-TM-SM, and high M r caldesmon. In situ hybridization revealed specific coexpression of ␣-TM-SM and high M r caldesmon in smooth muscle in developing embryos. These results suggest a common splicing mechanism for phenotype-dependent expression of tropomyosin and caldesmon isoforms in both visceral and vascular smooth muscle cells.It is important to elucidate the molecular mechanism of phenotypic modulation of smooth muscle cells (SMCs) 1 such as vasculogenesis, enterogenesis, atherosclerosis, hypertension, and leiomyogenic tumorigenesis. The SMCs are derived from mesodermal precursors, but the intracellular and extracellular factors determining the SMC lineage and its phenotype remain unclear. The search for molecular parameters indicating SMC phenotype is a first step in analyzing phenotypic modulation of SMCs. Several cytoskeletal and contractile proteins are such candidates. Among them, changes of actin (1, 2), caldesmon (CaD) (1, 3, 4), myosin heavy chain (5, 6), and vinculin/metavinculin (1, 7) isoforms are closely associated with phenotypic modulation of SMCs. Recent studies have focused on the gene regulation of such parameters (8 -14). In addition to these isoform changes, expression of ␣-smooth muscle actin (␣-SM actin) (15, 16), CaD (1, 3, 4), myosin heavy and light chains (5, 6, 17), meta-vinculin (1, 7), SM22, and calponin (18 -20) have been reported to be up-regulated during differentiation of SMCs, but down-regulated during dedifferentiation, suggesting the involvement of SMC phenotype-dependent transcriptional regulation in the SMC-specific parameter genes. In fact, the transcriptional machineries of ␣-SM actin (8, 9), CaD (12), myosin heavy chain (13), SM22 (21), and calponin (22) have been partially characterized.Tropomyosin (TM) is a predominant helical protein that binds to actin groves. Recent evidence suggests that Ca 2ϩ -dependent actin-myosin interaction in smooth and nonmuscle cells is controlled by myosin-and actin-linked dual regulation....

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mentioning

confidence: 99%

Coordinate Expression of α-Tropomyosin and Caldesmon Isoforms in Association with Phenotypic Modulation of Smooth Muscle Cells

Kashiwada¹,

Nishida²,

Hayashi³

et al. 1997

Journal of Biological Chemistry

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“…It is clear that the strength of the adjacent branch point also plays an important role in splice site selection (3,24). Moreover, studies on regulated alternative splicing have identified exonic splicing elements that facilitate the process of exon definition.…”

Section: Discussionmentioning

confidence: 99%

“…The progressive deletion of the polypyrimidine tract abolish lariat formation, spliceosome assembly, and consequently the splicing process (3,4), whereas increasing the length of the pyrimidine run can lead to improved efficiency of splicing in some systems (4).…”

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confidence: 99%

An Exonic Splicing Enhancer Offsets the Atypical GU-rich 3′ Splice Site of Human Apolipoprotein A-II Exon 3

Arrisi-Mercado

Romano

Muro

et al. 2004

Journal of Biological Chemistry

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Human apolipoprotein A-II (apoA-II) intron 2/exon 3 junction shows a peculiar tract of alternating pyrimidines and purines (GU tract) that makes the acceptor site deviate significantly from the consensus. However, apoA-II exon 3 is constitutively included in mRNA. We have studied this unusual exon definition by creating a construct with the genomic fragment encompassing the whole gene from apoA-II and its regulatory regions. Transient transfections in Hep3B cells have shown that deletion or replacement of the GU repeats at the 3 splice site resulted in a decrease of apoA-II exon 3 inclusion, indicating a possible role of the GU tract in splicing. However, a 3 splice site composed of the GU tract in heterologous context, such as the extra domain A of human fibronectin or cystic fibrosis transmembrane conductance regulator exon 9, resulted in total skipping of the exons. Next, we identified the exonic cis-acting elements that may affect the splicing efficiency of apoA-II exon 3 and found that the region spanning from nucleotide 87 to 113 of human apoA-II exon 3 is essential for its inclusion in the mRNA. Overlapping deletions and point mutations (between nucleotides 91 and 102) precisely defined an exonic splicing enhancer (ESEwt). UV cross-linking assays followed by immunoprecipitation with anti-SR protein monoclonal antibodies showed that ESEwt, but not mutated ESE RNA, was able to bind both alternative splicing factor/splicing factor 2 and SC35. Furthermore, overexpression of both splicing factors enhanced exon 3 inclusion. These results show that this protein-ESE interaction is able to promote the incorporation of exon 3 in mRNA and suggest that they can rescue the splicing despite the noncanonical 3 splice site.Pre-mRNA splicing is the process by which introns are removed and exons are joined together by a two-step trans-esterification reaction carried out by the spliceosome, a dynamic 60 S ribonucleoprotein particle (1). Formation of the spliceosome at particular splice junctions is triggered by recognition of the 5Ј splice site by the U1 small nuclear ribonucleoprotein and of the 3Ј splice site by U2AF followed by the U2 small nuclear ribonucleoprotein recognition of the branch point (2).The polypyrimidine tract is one of the important cis-acting sequences present in the 3Ј splice site of introns. The progressive deletion of the polypyrimidine tract abolish lariat formation, spliceosome assembly, and consequently the splicing process (3, 4), whereas increasing the length of the pyrimidine run can lead to improved efficiency of splicing in some systems (4).In vitro studies have demonstrated that the ability of polypyrimidine tracts to favor specific 3Ј splice site selection is not only determined by its length but also by its composition (4, 5). This feature coexists with a degree of flexibility in the specific sequence of a given tract.The human apolipoprotein A-II (apoA-II) 1 gene presents a peculiar arrangement of GT repeats within the polypyrimidine tract region at the intron 2/exon 3 junction that devia...

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“…In this case, the distance between the branch point in intron 2 and exon 2 is only 42 bases, which is too short to allow exon 2 to exon 3 splicing. The splice site choice between exons 2 and 3 results, at least partly, from the relative strength of the polypyrimidine tracts adjacent to the branch point [21]. However, they are not predominantly involved in skipping exon 9 in non M,-expressing cells.…”

Section: Discussionmentioning

confidence: 99%

Alternative Splicing of the Pyruvate Kinase M Gene in a Minigene System

Takenaka

Yamada

Lü

et al. 1996

European Journal of Biochemistry

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The MI-type and M,-type isozymes of pyruvate kinase are produced from a single gene by mutually exclusive use of exons 9 and 10. Selection of exon 10 generates the M, type, which occurs in most tissues, whereas the M, type is expressed by use of exon 9 only in skeletal muscle, heart and brain. We investigated the mechanism by which exon 10, but not exon 9 is selected in M,-expressing cells by transfecting minigenes containing exon 9 and/or exon 10 in cells and by analyzing the transcripts using reverse-transcriptase polymerase chain reaction. Deletion of the most conserved region in intron 8 did not affect selection of exon 10 in dRLh-84 cells, which express only the M, type. Exclusion of exon 10 from the minigene resulted in two major spliced products. One included correctly spliced exon 9 and the other skipped this exon. Similar splicing patterns were observed when these minigenes were transfected in hepatocytes which express the L type, but not M, or M, types. The 5' splice site but not the 3' splice site of exon 9 was found to be hardly recognized by the splicing machinery in dRLh-84 cells. Mutation of the 5' splice site sequence of exon 9 to that of exon 10 and vice versa did not change the splicing patterns. However, mutation of this site of exon 9 to a perfectly complementary sequence of U1 snRNA resulted in selection of exon 9 correctly spliced to exon 10. A 9-10 fusion exon (constructed by substitution of 68 bases of the 3' portion of exon 9 and 33 bases of the 5' portion of intron 9 for the corresponding regions of exon 10 and intron 10) was also correctly incorporated into a major product together with exon 10. Thus, we propose that exon 9 is not recognized in non MI-expressing cells due to the weak signal of its 5' splice site and that, although the 5' splicing signal of exon 10 also appears to be weak, this exon can be recognized in these cells because the 5' recognition signal may be relatively strengthened by cis-acting element(s) which may be present in the 3' portion of exon 9 and the 5' portion of intron 9 andlor the corresponding regions of exon 10 and intron 10.Keywords: pyruvate kinase M gene ; isozymes ; alternative splicing.In many animal genes, a single gene generates multiple isoforms. This is accomplished by alternative transcription and/or alternative splicing [I 1. Generally, these processes are regulated tissue specifically and developmentally. Pyruvate kinase provides a good example. This important glycolytic enzyme has four isoforms named the L, R, M, and M, types in mammals 12-51. They differ in primary structure, kinetic properties and tissue distribution. The M, type, which is considered to be the prototype, is the only form detected in early fetal tissues and is expressed in many adult tissues. This form is progressively replaced by the M I type in the skeletal muscle, heart and brain, by the L type in the liver, and by the R type in red blood cells during development or differentiation [6, 71. We demonstrated that these isozymes are encoded by two geneslhaploid genome ; the L ...

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Alpha-tropomyosin mutually exclusive exon selection: competition between branchpoint/polypyrimidine tracts determines default exon choice.

Cited by 148 publications

References 58 publications

Coordinate Expression of α-Tropomyosin and Caldesmon Isoforms in Association with Phenotypic Modulation of Smooth Muscle Cells

Coordinate Expression of α-Tropomyosin and Caldesmon Isoforms in Association with Phenotypic Modulation of Smooth Muscle Cells

An Exonic Splicing Enhancer Offsets the Atypical GU-rich 3′ Splice Site of Human Apolipoprotein A-II Exon 3

Alternative Splicing of the Pyruvate Kinase M Gene in a Minigene System

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