Alternative splicing of beta-tropomyosin pre-mRNA: multiple cis-elements can contribute to the use of the 5'- and 3'-splice sites of the nonmuscle/smooth muscle exon 6.
We previously found that the splicing of exon 5 to exon 6 in the rat beta-TM gene required that exon 6 first be joined to the downstream common exon 8 (Helfman et al., Genes and Dev. 2, 1627-1638, 1988). Pre-mRNAs containing exon 5, intron 5 and exon 6 are not normally spliced in vitro. We have carried out a mutational analysis to determine which sequences in the pre-mRNA contribute to the inability of this precursor to be spliced in vitro. We found that mutations in two regions of the pre-mRNA led to activation of the 3'-splice site of exon 6, without first joining exon 6 to exon 8. First, introduction of a nine nucleotide poly U tract upstream of the 3'-splice site of exon 6 results in the splicing of exon 5 to exon 6 with as little as 35 nucleotides of exon 6. Second, introduction of a consensus 5'-splice site in exon 6 led to splicing of exon 5 to exon 6. Thus, three distinct elements can act independently to activate the use of the 3'-splice site of exon 6: (1) the sequences contained within exon 8 when joined to exon 6, (2) a poly U tract in intron 5, and (3) a consensus 5'-splice site in exon 6. Using biochemical assays, we have determined that these sequence elements interact with distinct cellular factors for 3'-splice site utilization. Although HeLa cell nuclear extracts were able to splice all three types of pre-mRNAs mentioned above, a cytoplasmic S100 fraction supplemented with SR proteins was unable to efficiently splice exon 5 to exon 6 using precursors in which exon 6 was joined to exon 8. We also studied how these elements contribute to alternative splice site selection using precursors containing the mutually exclusive, alternatively spliced cassette comprised of exons 5 through 8. Introduction of the poly U tract upstream of exon 6, and changing the 5'-splice site of exon 6 to a consensus sequence, either alone or in combination, facilitated the use of exon 6 in vitro, such that exon 6 was spliced more efficiently to exon 8. These data show that intron sequences upstream of an exon can contribute to the use of the downstream 5'-splice, and that sequences surrounding exon 6 can contribute to tissue-specific alternative splice site selection.
Tsukahara
T
T
Cold Spring Harbor Laboratory, NY 11724.
Casciato
C
C
Helfman
D M
DM
D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D005091:Exons; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000378:metabolism*; D009132:Muscles / Q000378:metabolism; D009928:Organ Specificity / Q000235:genetics; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D014335:Tropomyosin / Q000235:genetics*
D012322:RNA Precursors; D014335:Tropomyosin