Splicing of Two Internal and Four Carboxyl-terminal Alternative Exons in Nonmuscle Tropomyosin 5 Pre-mRNA Is Independently Regulated during Development

Abstract

Four nonmuscle tropomyosin isoforms have been reported to be produced from the rat Tm5 gene by alternative splicing (Beisel, K. W., and Kennedy, J. E. (1994) Gene (Amst.) 145, 251-256). In order to detect additional isoforms that might be expressed from that gene, we used reverse transcriptase-polymerase chain reaction assays and evaluated the presence of all product combinations of two alternative internal exons (6a and 6b) and four carboxyl-terminal exons (9a, 9b, 9c, and 9d) in developing and adult rat brain. We identified five different combinations for exon 9 (9a ؉ 9b, 9a ؉ 9c, 9a ؉ 9d, 9c, and 9d), and the exon combinations 9a ؉ 9c and 9a ؉ 9d were previously unreported. Each of these combinations existed with both exon 6a and exon 6b. Thus, the rat brain generates at least 10 different isoforms from the Tm5 gene. Northern blot hybridization with alternative exon-specific probes revealed that these isoforms were also expressed in a number of different adult rat tissues, although some exons are preferentially expressed in particular tissues. Studies of regulation of the 10 different Tm5 isoform mRNAs during rat brain development indicated that no two isoforms are coordinately accumulated. Furthermore, there is a developmental switch in the use of exon 6a to exon 6b from embryonic to adult isoforms. TM5 protein isoforms show a differential localization in the adult cerebellum.Tropomyosins are rod-like proteins that are associated with actin filament in muscle and nonmuscle cells. Multiple isoforms of tropomyosins exist in both muscle and nonmuscle cells (1). In muscle cells, tropomyosins play a pivotal role in regulating the interaction between the actin and myosin filaments. The role of tropomyosins in nonmuscle cells is beginning to be more defined. They are thought to differentially affect the stability of actin filaments (2) and have been shown to be implicated in various cellular functions including the regulation of cell transformation (3-5), cytokinesis (6), motility (7,8), and morphogenesis (9 -13).Alternative splicing accounts for the majority of tropomyosin isoform diversity. In mammals, four tropomyosin genes have been identified, ␣-Tm f (14), ␤-Tm (15), Tm4 (16), and Tm5 (17). Except for the Tm4 gene, it is now clear that the other three rat tropomyosin genes contain two alternate promoters (exons 1a and 1b), two alternative internal exons (6a and 6b), and four alternative COOH-terminal exons (9a, 9b, 9c, and 9d). Therefore, the tropomyosin genes can each theoretically generate mRNAs for at least 16 isoforms. However, only 20 isoforms have been characterized from all four rat tropomyosin genes (14 -24). This suggests that some exon combinations either do not occur or are unique to cell types that have not yet been studied.In the case of the rat Tm5 gene, one muscle isoform and four nonmuscle isoforms have been reported to date. The muscle product of this gene is known as ␣-Tm s since it is preferentially expressed in slow-twitch skeletal muscle (25). The four nonmuscle isoforms have been termed T...

Specific Features of Neuronal Size and Shape Are Regulated by Tropomyosin Isoforms

“…Differentiation and morphogenesis in the brain and skeletal muscle are accompanied by extensive changes in Tm isoform expression (Gunning et al, 1990;Stamm et al, 1993;Weinberger et al, 1993Weinberger et al, , 1996Dufour et al, 1998). Although consistent with a direct role for tropomyosin in morphogenesis, no experiments have addressed the specificity of tro- pomyosin isoform function in this process.…”

“…Tms are derived from four highly conserved genes known as the ␣Tm fast , ␤Tm, ␥Tm (Tm5NM), and ␦Tm genes that, via alternative splicing, give rise to Ͼ40 isoforms (LeesMiller and Helfman, 1991;Dufour et al, 1998;Cooley and Bergtrom, 2001). Despite the well understood function of Tms in muscle where, together with the troponin complex, they regulate contraction in a calcium-dependent manner, little is known about their role in nonmuscle cells.…”

Gamma Tropomyosin Gene Products Are Required for Embryonic Development

“…Many nonmuscle isoforms are generated from the ␥Tm gene ( Fig. 1), and to date, 11 nonmuscle (NM1 to NM11) isoforms have been identified (12). The exact role for all of the 11 nonmuscle isoforms is yet to be determined.…”

Identification of a founder mutation in TPM3 in nemaline myopathy patients of Turkish origin

“…P05753, cDNA accession no. X04201; exon numbering as in Dufour et al 19 (b) The sequence chromatogram of the intron 9a -exon 9b boundary of TPM3. The WT (topmost), the sequence of a heterozygous carrier (on line two ; 'A' underlined is deleted from one of the alleles), the sequence of a homozygous patient (on line three).…”