RNA polymerase III transcription machinery and tRNA processing are regulated by the ubiquitin ligase Rsp5

Transfer RNA (tRNA) biogenesis in yeast involves synthesis of the primary transcript by RNA polymerase III (Pol III), followed by processing to remove 5' and 3' ends, further maturation, and export to the cytoplasm. In the present study, we found that both tRNA transcription and the initial processing of tRNA precursors are affected by the ubiquitin ligase Rsp5. We observed high levels of unprocessed primary tRNA transcripts in rsp5 mutants at elevated temperature, which were reduced upon the overexpression of RPR1, the catalytic subunit of RNase P. This observation suggests a role for Rsp5 in the maturation of 5' ends of tRNA precursors. Under the same conditions, in vivo labeling showed that the amount of newly synthesized tRNA decreased. Furthermore, we found that Rsp5 directly interacted with the Tfc3 subunit of the TFIIIC transcription factor, which is modified by ubiquitination. The inactivation of Rsp5 catalytic activity influenced the interaction between the general Pol III factors TFIIIB and TFIIIC and decreased the recruitment of TFIIIC to tRNA genes. These findings suggest that Rsp5 ligase is implicated in the control of Pol III transcription in yeast.