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

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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.