Nuclear RNA Surveillance in Saccharomyces Cerevisiae Requires a Three-Protein Polyadenylation Complex
Date of Award
Dissertation - Restricted
Doctor of Philosophy (PhD)
Effective turnover of many incorrectly processed RNAs, including hypomodified tRNAi Met, requires the TRAMP complex, which appends a short polyA tail to the RNA designated for decay that stimulates RNA degradation by the exosome. The TRAMP complex contains the polyA polymerase Trf4p, the RNA binding protein Air2p, and the DExH RNA helicase Mtr4p. I establish in this study that Trf4p-directed polyadenylation of hypomethylated tRNAi Met is required for its degradation, and Air2p confers RNA binding ability to Trf4p. The role of Mtr4p in RNA degradation processes involving the TRAMP complex has been unclear. Here I show through a genetic analysis that MTR4 is required for degradation but not for polyadenylation of hypomodified tRNAi Met. A suppressor of the trm6-504 mutation in the tRNA m1A58 methyltransferase (Trm6p/Trm61p), which causes a reduced level of tRNAi Met, was mapped to MTR4. This mtr4-20 mutation stabilizes hypomodified tRNAi Met in vivo but has no effect on TRAMP complex stability or polyadenylation activity in vivo or in vitro. In this study, I further show that purified recombinant Mtr4p displays RNA-dependent ATPase activity and separates partial RNA duplexes with a 3’ to 5’ polarity in an ATP or dATP-dependent fashion. The strand displacement and RNA stimulated ATPase activities are strongly reduced in the recombinant mutant Mtr4-20p, suggesting that these activities of Mtr4p are critical for degradation of polyadenylated hypomodified tRNAi Met. My results demonstrate that displacement of single strand RNAs from partial duplexes by Mtr4p requires the presence of a 3’-end single-stranded overhang that is longer than 1 nucleotide, and a RNA loading strand in the duplex. Additionally, the strand displacement rate of Mtr4p is relative to the overhang length. I also show that TRAMP exhibits better displacement activity than Mtr4p alone. These data indicate that the activity of Mtr4p might be regulated through polyadenylation by Trf4p-Air2p.