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

Detection of polyadenylated tRNA_i^Met in trm6-504 and trm6-504 suppressor mutants. Total RNA and poly(A)⁺ RNA were isolated from wild-type (Y200), trm6-504(Y190), trm6-504 trf4-20 (sup1), and trm6-504 rrp44-20(sup2) strains grown in YPD at 30°C as detailed in Materials and Methods. Total RNA (5 μg) and poly(A)⁺ RNA (2–4 μg) were separated on a 6% polyacrylamide (19:1) 8 Murea gel. Membrane-bound tRNA_i^Met was detected using a radiolabeled oligonucleotide JA11 followed by autoradiography.