Identification of two distinct intron elements involved in alternative splicing of beta-tropomyosin pre-mRNA.

The rat beta-tropomyosin gene encodes two isoforms, termed skeletal muscle beta-tropomyosin and fibroblast last tropomyosim 1 (TM-1), via an alternative RNA processing mechanism. The gene contains 11 exons. Exons 1-5 and exons 8 and 9 are common to all mRNAs expressed from the gene. Exons 6 and 11 are used in fibroblasts, as well as smooth muscle, whereas exons 7 and 10 are used only in skeletal muscle. In the present studies we focused on the mutually exclusive internal alternative splice choice involving exon 6 (fibroblast-type splice) and exon 7 (skeletal muscle-type splice). We have identified two distinct elements in the intron, upstream of exon 7, involved in splice site selection. The first element is comprised of a polypyrimidine tract located 89-143 nucleotides upstream of the 3' splice site, which specifies the location of the lariat branchpoints used, 144-153 nucleotides upstream of exon 7. The 3' splice site AG dinucleotide has no role in selection of these branchpoints. The second element is comprised of intron sequences located between the polypyrimidine tract and the 3' splice site of exon 7. It contains an important determinant in alternative splice site selection, because deletion of these sequences results in the use of the skeletal muscle-specific exon in nonmuscle cells. We propose that the use of lariat branchpoints located far upstream from a 3' splice site may be a general feature of some alternatively excised introns, reflecting the presence of regulatory sequences located between the lariat branch site and the 3' splice site. The data also indicate that alternative splicing of the rat beta-tropomyosin gene is regulated by a somewhat different mechanism from that described for rat alpha-tropomyosin gene and the transformer-2 gene of Drosophila melanogaster.

Alternative splicing of tropomyosin pre-mRNAs in vitro and in vivo.

A single rat gene encodes both fibroblast TM-1 and skeletal muscle beta-tropomyosin by an alternative RNA-processing mechanism. The gene contains 11 exons: Exons 1-5 and exons 8 and 9 are constitutive exons common to all mRNAs expressed from this gene; exons 6 and 11 are used in fibroblasts as well as smooth muscle; exons 7 and 10 are used exclusively in skeletal muscle. We have studied the internal alternative RNA splice choice (exons 6 and 7) of the rat tropomyosin 1 gene in vitro, using nuclear extracts obtained from HeLa cells. Use of alternative splice sites in vitro is dependent on the ionic conditions of the assay, and correct splicing occurs only under well-defined salt conditions. Splicing of exon 5 to exon 6 (fibroblast-type splice) and exon 5 to exon 7 (skeletal muscle-type splice) was dependent on precursors in which exon 6 or 7 was first joined to exon 8. The same patterns of alternatively spliced RNAs were formed when similar templates were introduced in HeLa cells by transfection. Thus, there appears to be an ordered pathway of splicing in which the internal alternatively spliced exons must first be joined to the downstream constitutive exon before they can be spliced to the upstream constitutive exon. The data are consistent with a model in which the critical event in alternative splicing occurs during the joining of exon 6 to exon 8 (fibroblast-type splice) or exon 7 to exon 8 (skeletal muscle-type splice).

Use of RNase H and primer extension to analyze RNA splicing.

A new method for the characterization of pre-mRNA splicing products is presented. In this method RNA molecules are hybridized to an oligodeoxynucleotide complementary to exon sequences upstream of a given 5' splice site, and the RNA strands of the resulting RNA:DNA hybrids are cleaved by RNase H. The cleaved RNAs are then subjected to primer extension using a 32P-labelled primer complementary to exon sequences downstream of an appropriate 3' splice site. Since the primer extension products all terminate at the site of RNase H cleavage, their lengths are indicative of the splice sites utilized. The method simplifies the study of the processing of complex pre-mRNAs by allowing the splicing events between any two exons to be analyzed. We have used this approach to characterize the RNAs generated by expression of the rat tropomyosin 1 (Tm 1) gene in various rat tissues and in cultured cells after transient transfection. The results demonstrate that this method is suitable for the analysis of alternative RNA processing in vivo.

Nonmuscle and muscle tropomyosin isoforms are expressed from a single gene by alternative RNA splicing and polyadenylation.

The molecular basis for the expression of rat embryonic fibroblast tropomyosin 1 and skeletal muscle beta-tropomyosin was determined. cDNA clones encoding these tropomyosin isoforms exhibit complete identity except for two carboxy-proximal regions (amino acids 189 to 213 and 258 to 284) and different 3'-untranslated sequences. The isoform-specific regions delineate the troponin T-binding domains of skeletal muscle tropomyosin. Analysis of genomic clones indicates that there are two separate loci in the rat genome that contain sequences complementary to these mRNAs. One locus is a pseudogene. The other locus contains a single gene made up of 11 exons and spans approximately 10 kilobases. Sequences common to all mRNAs were found in exons 1 through 5 (amino acids 1 to 188) and exons 8 and 9 (amino acids 214 to 257). Exons 6 and 11 are specific for fibroblast mRNA (amino acids 189 to 213 and 258 to 284, respectively), while exons 7 and 10 are specific for skeletal muscle mRNA (amino acids 189 to 213 and 258 to 284, respectively). In addition, exons 10 and 11 each contain the entire 3'-untranslated sequences of the respective mRNAs including the polyadenylation site. Although the gene is also expressed in smooth muscle (stomach, uterus, and vas deferens), only the fibroblast-type splice products can be detected in these tissues. S1 and primer extension analyses indicate that all mRNAs expressed from this gene are transcribed from a single promoter. The promoter was found to contain G-C-rich sequences, a TATA-like sequence TTTTA, no identifiable CCAAT box, and two putative Sp1-binding sites.