Takenaka M, Yamada K, Lu T, Kang R, Tanaka T, Noguchi T
Department of Nutrition and Physiological Chemistry, Osaka University Medical School, Japan.
Eur J Biochem. 1996 Jan 15;235(1-2):366-71. doi: 10.1111/j.1432-1033.1996.00366.x.
The M1-type and M2-type isozymes of pyruvate kinase are produced from a single gene by mutually exclusive use of exons 9 and 10. Selection of exon 10 generates the M2 type, which occurs in most tissues, whereas the M1 type is expressed by use of exon 9 only in skeletal muscle, heart and brain. We investigated the mechanism by which exon 10, but not exon 9 is selected in M2-expressing cells by transfecting minigenes containing exon 9 and/or exon 10 in cells and by analyzing the transcripts using reverse-transcriptase polymerase chain reaction. Deletion of the most conserved region in intron 8 did not affect selection of exon 10 in dRLh-84 cells, which express only the M2 type. Exclusion of exon 10 from the minigene resulted in two major spliced products. One included correctly spliced exon 9 and the other skipped this exon. Similar splicing patterns were observed when these minigenes were transfected in hepatocytes which express the L type, but not M1 or M2 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-84 cells. Mutation of the 5' splice site sequence of exon 9 to that of exon 10 and vice versa did not change the splicing patterns. However, mutation of this site of exon 9 to a perfectly complementary sequence of U1 snRNA resulted in selection of exon 9 correctly spliced to exon 10. 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 the corresponding regions of exon 10 and intron 10) was also correctly incorporated into a major product together with exon 10. Thus, we propose that exon 9 is not recognized in non M1-expressing cells due to the weak signal of its 5' splice site and that, although the 5' splicing signal of exon 10 also appears to be weak, this exon can be recognized in these cells because the 5' recognition signal may be relatively strengthened by cis-acting element(s) which may be present in the 3' portion of exon 9 and the 5' portion of intron 9 and/or the corresponding regions of exon 10 and intron 10.
丙酮酸激酶的M1型和M2型同工酶由单个基因通过互斥使用外显子9和10产生。选择外显子10产生M2型，其存在于大多数组织中，而M1型仅通过使用外显子9在骨骼肌、心脏和大脑中表达。我们通过在细胞中转染含有外显子9和/或外显子10的小基因，并使用逆转录酶聚合酶链反应分析转录本，研究了在表达M2的细胞中选择外显子10而非外显子9的机制。缺失内含子8中最保守的区域并不影响仅表达M2型的dRLh - 84细胞中外显子10的选择。从小基因中排除外显子10产生了两种主要的剪接产物。一种包含正确剪接的外显子9，另一种跳过了该外显子。当这些小基因转染到表达L型而非M1或M2型的肝细胞中时，观察到了类似的剪接模式。发现外显子9的5'剪接位点而非3'剪接位点在dRLh - 84细胞中很难被剪接机制识别。将外显子9的5'剪接位点序列突变为外显子10的序列，反之亦然，并没有改变剪接模式。然而，将外显子9的该位点突变为与U1 snRNA完全互补的序列导致正确剪接至外显子10的外显子9被选择。一个9 - 10融合外显子（通过将外显子9的3'部分的68个碱基和内含子9的5'部分的33个碱基替换为外显子10和内含子10的相应区域构建）也与外显子10一起正确地并入了一个主要产物中。因此，我们提出，由于其5'剪接位点信号较弱，外显子9在非M1表达细胞中不被识别，并且，尽管外显子10的5'剪接信号似乎也较弱，但该外显子在这些细胞中可以被识别，因为5'识别信号可能被存在于外显子9的3'部分和内含子9 的5'部分和/或外显子10和内含子10的相应区域中的顺式作用元件相对增强。
