Polyadenylation and degradation of hypomodified tRNAiMet.
Sujatha Kadaba (1), Annette Krecic (1), Tamyra Trice (1), Anna Krueger (2), Alan Hinnebusch (3), James Anderson (1)
(1) Biological Sciences, Marquette University, 530 n 15th St, Milwaukee, WI, 53233, United States;
(2) Department of Biology, Johns Hopkins University, Baltimore, MD 21218 USA;
(3) Laboratory of Gene Regulation and Development, National Institute of Child Health and Human Development, NIH, Bethesda, MD 20892 USA
Trm6p and Trm61p comprise the tRNA m1A58 methyltransferase in S. cerevisiae. Mutations in TRM6 result in dramatic reduction in the steady state tRNAiMet levels due to increased degradation. Extragenic suppressor analysis of a trm6-504 mutation has revealed that spontaneous mutations in TRF4 and RRP44 suppress trm6-504. Suppressors accumulate near wild type levels of tRNAiMet, suggesting that suppressor function influences tRNA instability. Trf4p possesses DNA and PolyA polymerase activities. Rrp44p is a component of the exosome, a multiprotein complex involved in RNA processing and turnover in the nucleus and cytoplasm. The nuclear and the cytoplasmic exosome complexes share core proteins but differ in their accessory components. Trf4p functions in conjunction with the nuclear exosome to degrade hypomodified pre-tRNAiMet. Rrp6p is a nonessential component of the nuclear exosome. A trm6-504 rrp6del mutant accumulates polyadenylated hypomodified tRNAiMet that is not observed in WT or rrp6del strains. The polyadenylated tRNAiMet increases in abundance, and it displays a wider length distribution in the trm6-504 rrp6del mutant overexpressing Trf4p, indicating that Trf4p stimulates polyadenylation of hypomodified tRNAiMet. Treatment of polyadenylated tRNAiMet with oligo-dT and RNaseH converts it to a unique species similar in size to pre-tRNAiMet. These data strongly support a model where hypomodified tRNAiMet is polyadenylated and subsequently degraded by the nuclear exosome.
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