Celeste Casciato

Cold Spring Harbor Laboratory

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David M. Helfman

Korea Advanced Institute of Science and Technology

Toshifumi Tsukahara

Japan Advanced Institute of Science and Technology

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Cold Spring Harbor Laboratory

Korea Advanced Institute of Science and Technology

Podiatry Institute

Sylvester Comprehensive Cancer Center

University of Miami

Cancer Institute (WIA)

University of California, San Diego

University of Iowa

University of Arizona

Stony Brook University

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Alternative splicing of β-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

Nucleic Acids Research (1994)

Toshifumi Tsukahara Celeste Casciato David M. Helfman

We previously found that the splicing of exon 5 to exon 6 in the rat β-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.

Splice site mutation

Exon shuffling

Exon trapping

splice

Polypyrimidine tract

Precursor mRNA

Exonic splicing enhancer

Minigene

Exon skipping

10.1093/nar/22.12.2318

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