tRNA editing in S. cerevisiae: Isolation and Identification of the responsible nucleotidyltransferase

在后生动物的线粒体基因组中，一些tRNA基因与位于同一链上的相邻下游tRNA基因重叠多达6个核苷酸。在相应的多顺反子前体的加工过程中，下游的tRNA作为一个完整的分子被释放，而上游的tRNA缺少重叠位置，因此在其3‘端被截断。RNA编辑反应修复这些缺失的位置，并通过核苷酸掺入完成tRNA。有趣的是，当被引入酿酒酵母中时，这种截短的tRNA也得到了恢复，尽管这种生物不携带相应的内源tRNA作为编辑底物。显然，酵母携带一种混杂核苷酸，其活性可以接受这些截短的tRNAs作为底物，并将缺失的核苷酸添加到3‘端。这些数据支持RNA编辑通过招募预先存在的和混杂的核苷酸插入活动而进化的假说。我们设计了一系列补充方法，使用酵母菌的筛选程序和生化酶分析，将允许分离这种活性。此外，底物特异性、错误率和该活性恢复其他tRNA(tRNA修复)的能力将被确定。这些结果将为探讨RNA编辑事件的进化起源提供重要的贡献。

In the mitochondrial genomes of metazoans, some tRNA genes overlap by up to six nucleotides with the neighboring downstream tRNA gene located on the same strand. In the processing of the corresponding polycistronic precursor, the downstream tRNA is released as a complete molecule, while the upstream tRNA is missing the overlapping positions and is therefore truncated at its 3’- end. An RNA editing reaction restores these missing positions and completes the tRNA by nucleotide incorporation. Interestingly, when introduced into S. cerevisiae, such a truncated tRNA is likewise restored, although this organism does not carry corresponding endogenous tRNAs as editing substrates. Obviously, yeast carries a promiscuous nucleotide incorporating activity that accepts these truncated tRNAs as substrates and adds missing nucleotides to the 3’-end. These data support the hypothesis of the evolution of RNA editing by recruitment of a pre-existing and promiscuous nucleotide inserting activity. We have designed a series of complementary approaches using screening procedures in yeast and biochemical enzyme analyses that will allow the isolation of this activity. Furthermore, substrate specificity, error rate and the ability of this activity to restore other tRNAs (tRNA repair) will be determined. The results will provide an important contribution to the discussion about the evolutionary origin of RNA editing events.