Kawahara T, Yanagi H, Yura T, Mori K
HSP Research Institute, Kyoto, Japan.
J Biol Chem. 1998 Jan 16;273(3):1802-7. doi: 10.1074/jbc.273.3.1802.
Accumulation of unfolded proteins in the endoplasmic reticulum (ER) activates an intracellular signaling pathway from the ER to the nucleus, termed the unfolded protein response. We and others recently identified transcription factor Hac1p/Ern4p responsible for the response in Saccharomyces cerevisiae and found that Hac1p expression is controlled by the regulated splicing of HAC1 mRNA. Walter and co-workers (Sidrauski, C., Cox, J. S., and Walter, P. (1996) Cell, 87, 405-413) further showed that the splicing requires tRNA ligase but not spliceosome. In this report, we carried out mutational analysis of HAC1 mRNA and revealed several unique features of the splicing. First, a mutation or deletion of the branchpoint-like sequence present in HAC1 intron did not affect the splicing. Second, cleavage of the splice sites was sequence-specific and thus completely blocked by some point mutations introduced at the 5' or 3' splice site. Third, cleavage of the 5' and 3' splice sites could occur independently as judged by the nature of splicing intermediates accumulated. Fourth, swapping the nucleotide sequences of the 5' and 3' splice sites inhibited the ligation but not the cleavage step. We conclude that signaling from the ER activates putative endonucleases that can carry out sequence-specific cleavage of the splice sites in a random order.
内质网（ER）中未折叠蛋白的积累激活了一条从内质网到细胞核的细胞内信号通路，称为未折叠蛋白反应。我们和其他研究人员最近在酿酒酵母中鉴定出负责该反应的转录因子Hac1p/Ern4p，并发现Hac1p的表达受HAC1 mRNA可变剪接的调控。Walter及其同事（Sidrauski, C., Cox, J. S., and Walter, P. (1996) Cell, 87, 405 - 413）进一步表明，这种剪接需要tRNA连接酶而不是剪接体。在本报告中，我们对HAC1 mRNA进行了突变分析，并揭示了剪接的几个独特特征。首先，HAC1内含子中存在的分支点样序列的突变或缺失并不影响剪接。其次，剪接位点的切割具有序列特异性，因此在5'或3'剪接位点引入的一些点突变会完全阻断切割。第三，根据积累的剪接中间体的性质判断，5'和3'剪接位点的切割可以独立发生。第四，交换5'和3'剪接位点的核苷酸序列会抑制连接步骤，但不会抑制切割步骤。我们得出结论，内质网发出的信号激活了假定的核酸内切酶，这些酶可以以随机顺序对剪接位点进行序列特异性切割。
