Metal ion catalysis during the exon-ligation step of nuclear pre-mRNA splicing: Extending the parallels between the spliceosome and group II introns
Metal ion catalysis during the exon-ligation step of nuclear pre-mRNA splicing: Extending the parallels between the spliceosome and group II introns
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DOI：
10.1017/s1355838200992069
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发表时间：
2000-02-01
期刊：
影响因子：
4.5
通讯作者：
Piccirilli, JA
中科院分区：
文献类型：
--
作者：
Gordon, PM;Sontheimer, EJ;Piccirilli, JA
剪接体和II组内含子自我剪接的核前体mRNA剪接的机制分析提供了深入了解剪接的催化策略和不同剪接系统之间的进化关系。我们先前表明核前体mRNA的3'剪接位点处的3'-硫取代对剪接没有影响。我们现在报告，当使用双分子外显子连接测定监测剪接的第二步时，核前体mRNA的3'剪接位点处的3'-硫取代引起金属特异性的转换。这表明剪接体在剪接的第二步期间使用催化金属离子来稳定3 '-氧阴离子离去基团，如先前针对第一步所示。顺式剪接条件下缺乏金属特异性开关表明，剪接两个步骤之间的限速构象变化可能会掩盖随后的化学步骤和金属特异性开关。作为第二组内含子，一个真正的核酶，使用相同的催化策略剪接，我们的研究结果加强的论点，剪接体是一种RNA催化剂，共享一个共同的分子祖先与第二组内含子。
Mechanistic analyses of nuclear pre-mRNA splicing by the spliceosome and group II intron self-splicing provide insight into both the catalytic strategies of splicing and the evolutionary relationships between the different splicing systems. We previously showed that 3'-sulfur substitution at the 3' splice site of a nuclear pre-mRNA has no effect on splicing, We now report that 3'-sulfur substitution at the 3' splice site of a nuclear pre-mRNA causes a switch in metal specificity when the second step of splicing is monitored using a bimolecular exon-ligation assay. This suggests that the spliceosome uses a catalytic metal ion to stabilize the 3'-oxyanion leaving group during the second step of splicing, as shown previously for the first step. The lack of a metal-specificity switch under cis splicing conditions indicates that a rate-limiting conformational change between the two steps of splicing may mask the subsequent chemical step and the metal-specificity switch. As the group II intron, a true ribozyme, uses identical catalytic strategies for splicing, our results strengthen the argument that the spliceosome is an RNA catalyst that shares a common molecular ancestor with group II introns.