MULTIPLE ROLES FOR U6 SNRNA IN THE SPLICING PATHWAY
MULTIPLE ROLES FOR U6 SNRNA IN THE SPLICING PATHWAY
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DOI：
10.1101/gad.4.12b.2264
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发表时间：
1990-12-01
影响因子：
10.5
通讯作者：
GUTHRIE, C
中科院分区：
文献类型：
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作者：
MADHANI, HD;BORDONNE, R;GUTHRIE, C
U6是五个剪接体rna中最高度保守的。它通过广泛的碱基配对相互作用与U4相关联，这种相互作用在剪接的第一个溶核步骤之前立即被破坏。有人提出，该事件通过揭开U6来激活催化。使用掺杂合成和定点诱变的组合在U6中产生点突变，我们现在已经确定了3个结构域的12个位置，在这些位置上单核苷酸替换或缺失导致致命或温度敏感的表型。生化分析表明，这些突变体大多数保留了组装成U4/U6和U4/U5/U6 snRNPs的能力。值得注意的是，尽管在U6中与U4碱基对的三个位置发生突变是致命的，但U4中互补残基的突变是完全可行的。此外，恢复碱基配对的U4补偿性突变不能抑制U6突变的表型。这证明了U6独立于它在碱基配对中的作用。值得注意的是，在两种酵母菌中发现的三个基本区域中，有两个与内含子插入点对应。其中一个位点的温度敏感突变在体外剪接的第二步中是有缺陷的。
U6 is the most highly conserved of the five spliceosomal RNAs. It is associated with U4 by an extensive base-pairing interaction, which is disrupted immediately prior to the first nucleolytic step of splicing. It has been proposed that this event activates catalysis by unmasking U6. Using a combination of doped synthesis and site-directed mutagenesis to generate point mutations in U6, we have now identified 12 positions, in three domains, at which single nucleotide substitutions or deletions result in lethal or temperature-sensitive phenotypes. Biochemical analysis demonstrates that most of these mutants retain the ability to assemble into U4/U6 and U4/U5/U6 snRNPs. Notably although mutations at three positions in U6 that base-pair with U4 are lethal, mutations in the complementary residues in U4 are fully viable. Furthermore, compensatory mutations in U4 that restore base-pairing fail to suppress the phenotypes of the U6 mutations. This demonstrates a function for U6 independent of its role in base-pairing. Remarkably, two of the three essential regions in U6 identified genetically correspond to intron insertion points in two yeast species. A temperature-sensitive mutation at one of these sites is defective in the second step of splicing in vitro.