Role of transfer RNA post-transcriptional modification on translational misreading errors in the yeast Saccharomyces cerevisiae

Abstract

Post-transcriptional modification of tRNAs modulates the behavior of tRNAs in several ways. Anticodon loop modifications alter details of codon-anticodon interactions either stabilizing or destabilizing interactions with the codon. These modifications can alter the rate of cognate codon recognition and the frequency of misreading of near-cognate codons. Modifications outside the anticodon stem loop have been shown to alter the stability of the tRNA to degradation (Alexandrov et al., 2006). I am interested in whether these second sets of modifications also modulate misreading errors. This study aims to use S. cerevisiae as a system to understand the role of modification of the body of tRNA. I have exploited a set of misreading reporter systems developed in our laboratory to measure the effect of in vivo frequency of errors due to the lack of modifications to the core region of the tRNA. Since the modifications are distant from the anticodon loop their effect on misreading suggests that they stabilize the three-dimensional structure of the tRNA to increase fidelity. I present data showing that in the presence of several modification mutations the frequency of misreading either increases or decreases. I showed loss of various posttranscriptional modifications affects first position U?U, second position U?G, and wobble position U?U and U?C mismatches by tRNALys UUU and tRNAGlu UUC. These results support the idea that the tRNA body modifications are important for regulating decoding during translation. Next, I showed that the stability of tRNAs is not affected by the lack of nonessential core modifications at the permissive temperature. The northern blotting of the wild-type and mutant strains lacking the modification showed that the abundance of tRNAs is not reduced in the absence of body modification. These data were also confirmed with met22? which prevents the rapid tRNA decay pathway, and the misreading frequency did not go back to normal level in the met22? double mutants. Also, I evaluated the aminoacylation status of the tRNAs lacking body modification and report that the level of aminoacylation did not decrease in the absence of nonessential body modification in the permissive temperature. Finally, I studied the retrograde transport of hypomodified tRNAs to the nucleus, to analyze all the possible pathways by which the deletion of body modification may affect the availability of tRNA. My results showed that lack of mtr10?, the retrograde import receptor, had little to no effect on the frequency of misreading errors.