Nuclear RNA surveillance in Saccharomyces cerevisiae: Trf4p-dependent polyadenylation of nascent hypomethylated tRNA and an aberrant form of 5S rRNA

Trf4p and Trf5p have overlapping functions in the degradation of hypomethylated tRNA_i^Met. (A) Yeast strains wild type (Y200), trm6-504 (Y190), trm6-504 trf4-20 (sup1), and trm6-504 trf5Δ were grown to saturation in YPD and serial 10-fold dilutions were plated onto YPD plates and incubated at 26°C or 36°C for 3 d and 2 d, respectively. (B) The same strains from A transformed with empty vector (WT, trm6-504, and trm6-504 trf5Δ) or a high-copy number plasmid containing TRF5 (WT, trm6-504, and trm6-504 trf5Δ + hcTRF5) were streaked to synthetic complete media lacking uracil and incubated at 30°C and 36°C for 3 d. (C) Northern hybridization of total RNA (3 μg) and poly(A)⁺ RNA (4–5 μg) isolated from wild-type (Y200), trm6-504 trf4Δ and trm6-504 trf4Δ + hcTRF5. The blot was hybridized with a radiolabeled oligonucleotide (JA11) to detect tRNA_i^Met.