Figure 1.

Aberrant pre-rRNAs generated in the absence of Top1 are targeted for degradation by TRAMP and exosome complexes. (A) Schematic representation of a 35S rRNA gene in S. cerevisiae. Pre-rRNA processing sites A0, A1, D, A2, A3, B1, C2, C1, B2, and B0 are labeled. Probes used in Northern analyses are indicated by bars. The 35S, 20S, 27SA, and 27SB pre-rRNAs are depicted by solid arrows. Truncated pre-rRNA fragments generated in the absence of Top1 are depicted by dotted arrows and are labeled 1–9. Numbers of nucleotides along the rDNA unit relative to the start of transcription (0) are indicated. The pre-rRNA processing pathway is depicted in Supplemental Figure S1. (B–E) Polyadenylated pre-rRNA fragments accumulate in double top1Δ rrp6Δ mutants. Total RNA was extracted from wild-type (WT), rrp6Δ, top1Δ, and top1Δ rrp6Δ strains grown at 25°C. Polyadenylated RNAs were purified and resolved alongside total RNAs on a 1.2% glyoxal-agarose gel. Pre-rRNA fragments were detected by Northern analysis: probe 033, hybridizing at +278 in 5′-ETS (B); probe 004, hybridizing upstream of site A2 (C); and probe 003, hybridizing between A2 and A3 (D). (E) As controls for poly(A)⁺ RNA specificity, 25S and 18S rRNA and PGK1 mRNA were analyzed. (F,G) TRAMP participates in pre-rRNA surveillance in top1Δ mutants. Total RNA was extracted from wild-type and single and double top1Δ, trf4Δ, and rrp6Δ strains grown at 25°C and analyzed using probe 130 (hybridizes in the 5′-ETS upstream of probe 033) and 003. F and G are from two independent experiments. (Bottom panels) SCR1 was used as a loading control. Intact pre-rRNAs and truncated pre-rRNA fragments are labeled. “(18S)” and “(25S)” indicate the positions of migration of 18S and 25S rRNAs. Probe names are bracketed. The 23S RNA is an aberrant pre-rRNA processing intermediate extending from +1 to site A3, shown previously to be polyadenylated and targeted for degradation by TRAMP and Rrp6 (Dez et al. 2006).