ALTERNATIVE SPLICING OF THE PYRUVATE-KINASE-M GENE IN A MINIGENE SYSTEM

The M(1)-type and M(2)-type isozymes of pyruvate kinase are produced f rom a single gene by mutually exclusive use of exons 9 and 10. Selecti on of exon 10 generates the M(2) type, which occurs in most tissues, w hereas the M(1) type is expressed by use of exon 9 only in skeletal mu scle, heart and brain. We investigated the mechanism by which exon 10, but not exon 9 is selected in M(2)-expressing cells by transfecting m inigenes containing exon 9 and/or exon 10 in cells and by analyzing th e transcripts using reverse-transcriptase polymerase chain reaction. D eletion of the most conserved region in intron 8 did not affect select ion of exon 10 in dRLh-84 cells, which express only the M(2) type, Exc lusion of exon 10 from the minigene resulted in two major spliced prod ucts. One included correctly spliced exon 9 and the other skipped this exon. Similar splicing patterns were observed when these minigenes we re transfected in hepatocytes which express the L type, but not M(1) o r M(2) types. The 5' splice site but not the 3' splice site of exon 9 was found to be hardly recognized by the splicing machinery in dRLh-s4 cells, Mutation of the 5' splice site sequence of exon 9 to that of e xon 10 and vice versa did not change the splicing patterns. However, m utation of this site of exon 9 to a perfectly complementary sequence o f U1 snRNA resulted in selection of exon 9 correctly spliced to exon 1 0. A 9-10 fusion exon (constructed by substitution of 68 bases of the 3' portion of exon 9 and 33 bases of the 5' portion of intron 9 for th e corresponding regions of exon 10 and intron 10) was also correctly i ncorporated into a major product together with exon 10. Thus, we propo se that exon 9 is not recognized in non M(1)-expressing cells due to t he weak signal of its 5' splice site and that, although the 5' splicin g signal of exon 10 also appears to be weak, this exon can be recogniz ed in these cells because the 5' recognition signal may be relatively strengthened by cis-acting element(s) which may be present in the 3' p ortion of exon 9 and the 5' portion of intron 9 and/or the correspondi ng regions of exon 10 and intron 10.