Polyadenylation of Rnt1p cleavage intermediates requires Trf4p. (A) Analysis of the polyadenylation status of the U3 and U4 cleavage intermediates in trf4Δ-derived strains. The membrane was also hybridized with the RPL18A probe to test for equal loading in the poly(A)+RNA lanes. (B) 3′-Extended forms of the U4 snRNA accumulate in the absence of Trf4p. A membrane with total RNAs extracted from wild-type, trf4Δ, or rrp6Δ strains was hybridized with the U4 probe. The brace indicates the 3′-extended forms that accumulate in the absence of Trf4p or Rrp6p. (C) Analysis of the polyadenylation status of the RPL18A and snR40 cleavage intermediates in trf4Δ-derived strains. Legends as in Figure 1 ▶.

Polyadenylation of Rnt1p cleavage intermediates is influenced by Trf5p. (A) Analysis of the polyadenylation status of the U3 and U4 cleavage intermediates in trf5Δ-derived strains. The membrane was also hybridized with the RPL18A probe to test for equal loading in the poly(A)+RNA lanes. (B) Analysis of the polyadenylation status of the RPL18A and snR40 cleavage intermediates in trf5Δ-derived strains. Legends as in Figure 1 ▶. (C) The reduction of polyadenylated cleavage intermediates observed in the rrp6Δ trf5Δ double deletion strain for the U3 and U4 RNAs is reproducible. “A” and “B” represent RNAs extracted from two independent clones carrying the rrp6Δ trf5Δ double deletion; “1” and “2” represent two independent RNA preparations.

Analysis of the in vivo polyadenylation status of Rnt1p cleavage products and primary precursors. (A) Schematic representation of the primary precursors of the U4 snRNA, the U3 and snR40 snoRNAs, and the RPL18A and RPS22B mRNAs. Boxes represent mature or exonic sequences. Arrows represent the Rnt1p cleavage sites. Black lines with asterisks represent the location of the oligonucleotide probes used. (B) 3′-Processing intermediates and unprocessed precursors of the U3 snoRNA and of the U4 snRNA are polyadenylated. Total RNAs (T) or polyadenylated RNAs selected by oligo-dT affinity (P) extracted from the indicated strains were loaded on acrylamide gels, transferred to nylon membranes, and probed with oligonucleotides hybridizing to the mature sequences of the U3 snoRNA or U4 snRNA. Species with arrowheads represent the species that result from Rnt1p cleavage. The Arginine tRNA tR(UCU) was used as a negative control to test for contamination of purified polyadenylated RNAs by total RNAs. (C) The upstream degradation intermediate of the snR40 box C/D snoRNA is polyadenylated. Legends as in B. The probe used hybridizes against the 5′-extension of snR40 (see A); therefore, the mature snR40 snoRNA is not detected. (D) Degradation intermediates of the RPL18A and RPS22B unspliced pre-mRNAs are polyadenylated. Legends as in B. Probes used hybridize against the 5′-exon sequences of RPL18A or RPS22B. The same cleaved intermediate was detected by using an intronic oligonucleotide probe in the case of RPS22B (data not shown). For RPS22B, the unspliced precursor is not included in the gel.