Gordon P M, Sontheimer E J, Piccirilli J A
Department of Biochemistry and Molecular Biology, Howard Hughes Medical Institute, The University of Chicago, Illinois 60637, USA.
RNA. 2000 Feb;6(2):199-205. doi: 10.1017/s1355838200992069.
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.
对剪接体介导的细胞核前体mRNA剪接以及II类内含子自我剪接的机制分析，为剪接的催化策略以及不同剪接系统之间的进化关系提供了深入见解。我们之前表明，细胞核前体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法监测剪接的第二步时，细胞核前体mRNA的3'剪接位点处的3'-硫取代会导致金属特异性的转变。这表明剪接体在剪接的第二步中使用催化金属离子来稳定3'-氧阴离子离去基团，正如之前在第一步中所显示的那样。在顺式剪接条件下缺乏金属特异性转变表明，剪接两步之间限速的构象变化可能掩盖了随后的化学步骤和金属特异性转变。由于II类内含子是一种真正的核酶，采用相同的催化策略进行剪接，我们的结果强化了这样的观点，即剪接体是一种与II类内含子具有共同分子祖先的RNA催化剂。
