Alternative splicing of a human alpha-tropomyosin muscle-specific exon: identification of determining sequences.

Abstract

The human ac-tropomyosin gene hTM.m has two mutually exclusive versions of exon 5 (NM and SK), one of which is expressed specifically in skeletal muscle (exon SK). A minigene construct expresses only the nonmuscle (NM) isoform when transfected into COS-1 cells and both forms when transfected into myoblasts. Twenty-four mutants were produced to determine why the SK exon is not expressed in COS cells. The results showed that exons NM and SK are not in competition for splicing to the flanking exons and that there is no intrinsic barrier to splicing between the exons. Instead, exon SK is skipped whenever there are flanking introns. Splicing of exon SK was induced when the branch site sequence 70 nucleotides upstream of the exon was mutated to resemble the consensus and when the extremities of the exon itself were changed to the corresponding NM sequence. Precise swaps of the NM and SK exon sequences showed that the exon sequence effect was dominant to that of intron sequences. The mechanism of regulation appears to be unlike that of other tropomyosin genes. We propose that exclusion of exon SK arises because its 3' splicing signals are weak and are prevented by an exon-specific repressor from competing for splice site recognition.Alternative splicing of pre-mRNA produces different isoforms of mRNA from a single gene, often resulting in the production of multiple protein isoforms. In many cases, the proportions of the various isoforms alter during cell differentiation or development. Mutually exclusive splicing, whereby only one of two adjacent exons is incorporated between common flanking exons, is associated particularly with transcripts which are expressed primarily in muscle cells or which exhibit muscle-specific switches in the exon selected. This pattern of splicing raises three principal problems: (i) the two alternative exons are not usually spliced to each other, and where the process appears to be regulated, (ii) one exon is preferred in most cell types but (iii) the alternative exon is incorporated in the specific tissue.This pattern of splicing has been studied most intensively in two tropomyosin gene systems: the 5'-proximal exons 2 and 3 of rat a-tropomyosin, in which exon 2 is incorporated specifically in smooth muscle (39), and the more central exons 6 and 7 (or 6A and 6B) of the 3-tropomyosin homologs in rats and chickens, in which the 3' most of the two exons is incorporated in skeletal muscle (19,24,26). The results of these studies do not address all of the issues described above, but perhaps surprisingly, it is clear that mutually exclusive splicing is not determined by a common mechanism. In the case of the rat a-tropomyosin gene, it appears that exons 2 and 3 do not splice together because the long polypyrimidine tract of exon 3 forces the branch site too close to the 5' splice site of exon 2 (39). The default exon 3 appears to be selected because both exons are in competition for splicing to exon 1, but exon 3 is preceded by a more favorable polypyrimidine tract (32); sequences near or within ...

A splicing enhancer in the human fibronectin alternate ED1 exon interacts with SR proteins and stimulates U2 snRNP binding.

“…These results suggest that the SE element is not involved in the formation of essential secondary structures with other ED 1 exon sequences. Our observations contrast with the usual characteristics of mammalian exon sequences, as manipulations introduced into almost any region of other alternate exons provoked important changes in exon splicing in vivo and, when tested, in vitro (Hampson et al 1989;Streuli and Saito 1989;Libri et al 1990;Black 1991;Tacke and Goridis 1991;Black 1992;Cooper 1992;Cote et al 1992;Graham et al 1992). Thus, the SE element of the fibronectin ED 1 exon is rather exceptional among mammalian exon elements in its ability to efficiently promote 3' splice site recognition even following considerable molecular surgery on flanking exon sequences.…”

“…Several studies testify to the importance of exon sequences in splice site selection. Some exon sequences repress the use of adjacent splice sites (Gallego et al 1992;Nemeroff et al 1992;Siebel et al 1992)while other exon elements act positively by stimulating splicing (Reed and Maniatis 1986;Hampson et al 1989;Streuli and Saito 1989;Libri et al 1990;Cooper 1992;Cote et al 1992;Graham et al 1992). Recently, purine-rich elements acting as splicing enhancers have been identified in two m a m m a l i a n alternate exons (Watakabe et al 1993;Xu et al 1993).…”

Presence of Exon Splicing Silencers within Human Immunodeficiency Virus Type 1 tat Exon 2 and tat-rev Exon 3: Evidence for Inhibition Mediated by Cellular Factors

“…Recent reports have shown that the SR proteins selectively bind to purine-rich splicing elements present in cellular exons (30,49,50). There are also several examples of negative-acting exon splicing elements (2,4,18,45,55). To date, factors interacting with negative-acting exon splicing elements affecting alternative 3Ј splice site usage in metazoan cells have not yet been reported.…”

Multiple Interdependent Sequence Elements Control Splicing of a Fibroblast Growth Factor Receptor 2 Alternative Exon

“…The avian troponin T gene contains an optional exon spliced in the embryonic but not the adult heart (41,54). Alpha-and beta-tropomyosin genes contain pairs of mutually exclusive internal exons spliced with strict tissue specificity (5,14,24,25,32). The pre-mRNA encoding calcitonin and calcitonin gene-related peptide contains six exons, one of which (exon 4) is skipped in a few cell types, including neuronal cells.…”