A Sequential splicing mechanism promotes selection of an optimal exon by repositioning a downstream 5' splice site in preprotachykinin pre-mRNA.
A Sequential splicing mechanism promotes selection of an optimal exon by repositioning a downstream 5' splice site in preprotachykinin pre-mRNA.
复制标题
顺序剪接机制通过重新定位前原速激肽前体 mRNA 中的下游 5 剪接位点来促进最佳外显子的选择。
DOI：
10.1101/gad.4.7.1172
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发表时间：
1990
影响因子：
10.5
通讯作者：
Grabowski,PJ
中科院分区：
文献类型：
--
作者：
Nasim,FH;Spears,PA;Hoffmann,HM;Kuo,HC;Grabowski,PJ
为了探索选择性剪接的结构基础，我们分析了前体mRNA的剪接，该前体mRNA包含来自前体速激肽原基因的可选外显子E4。该基因通过共同前mRNA的选择性剪接编码P物质和相关速激肽肽。我们已经表明，前速激肽原前体mRNA的选择性剪接发生的优先跳过可选的E4。将E4掺入最终剪接的RNA的竞争机制受到对紧邻上游间插序列（IVS）IVS 3的剪接的初始阻断的限制。这种阻断通过顺序剪接来缓解，其中直接下游的IVS 4首先被去除。由第一剪接事件引起的结构变化直接负责IVS 3剪接的激活。这种结构重排用E5及其相邻的IVS 5序列取代IVS 4序列。为了确定这种结构变化如何促进IVS 3剪接，我们询问了什么结构变化将恢复IVS 3剪接缺陷突变体的活性。通过将E4的5'剪接位点转化为精确一致匹配的GUAAGU的2-核苷酸取代观察到最显著的效果。单独的外显子5序列被发现不促进剪接时，存在于一个或多个拷贝。然而，当将含有GUAAGU的IVS 5的15个核苷酸片段插入到杂交外显子片段E4 E5的下游的剪接缺陷突变体中时，剪接活性被恢复。奇怪的是，腺病毒的72个核苷酸的L2外显子，没有其相关的5'剪接位点，当与E4并置时激活剪接。模型的激活剪接的RNA结构的变化进行了讨论。
To explore the structural basis of alternative splicing, we have analyzed the splicing of pre-mRNAs containing an optional exon, E4, from the preprotachykinin gene. This gene encodes substance P and related tachykinin peptides by alternative splicing of a common pre-mRNA. We have shown that alternative splicing of preprotachykinin pre-mRNA occurs by preferential skipping of optional E4. The competing mechanism that incorporates E4 into the final spliced RNA is constrained by an initial block to splicing of the immediate upstream intervening sequence (IVS), IVS3. This block is relieved by sequential splicing, in which the immediate downstream IVS4 is removed first. The structural change resulting from the first splicing event is directly responsible for activation of IVS3 splicing. This structural rearrangement replaces IVS4 sequences with E5 and its adjacent IVS5 sequences. To determine how this structural change promoted IVS3 splicing, we asked what structural change(s) would restore activity of IVS3 splicing-defective mutants. The most significant effect was observed by a 2-nucleotide substitution that converted the 5' splice site of E4 to an exact consensus match, GUAAGU. Exon 5 sequences alone were found not to promote splicing when present in one or multiple copies. However, when a 15-nucleotide segment of IVS5 containing GUAAGU was inserted into a splicing-defective mutant just downstream of the hybrid exon segment E4E5, splicing activity was recovered. Curiously, the 72-nucleotide L2 exon of adenovirus, without its associated 5' splice site, activates splicing when juxtaposed to E4. Models for the activation of splicing by an RNA structural change are discussed.