Trf4p dependent polyadenylation of 3′ end truncated 5S rRNA. (A) A schematic diagram of 5S rDNA showing the coding region (filled box) and the 5′ and 3′ intergenic regions (lines). The approximate positions (not to scale) complementary to the oligonucleotide probes used in this study are shown as lines beneath the coding region. (B) Total RNA (5 μg) isolated from wild type (F39), rrp6Δ (F23), rrp44-20 (Y303), trf4Δ (F22), and trm6-504/trf4Δ (Y301) strains grown at 30°C was used for Northern hybridization analysis. The membrane was probed with JA99 to detect mature 5S rRNA, JA133 to detect 5S rRNA containing the first 20 nt of 5S rRNA and JA134 to detect 5S rRNA containing the last 20 nt. The 5S rRNA was visualized by autoradiography. (C) Total RNA (5 μg, lanes 1–5) and poly(A)+ RNA (2–4 μg, lanes 6–10) isolated from the same strains described in B was used for Northern hybridization analysis using probes JA133 (top) and JA134 (bottom). Indicated are the migration positions of the mature 5S rRNA, 3′-end truncated 5S rRNA, and poly(A)+ 5S rRNA, as well as the probe used for each hybridization.
