Mtr4p connects polyadenylation to degradation. Depletion of Mtr4p by growth of the GAL∷mtr4 strains on glucose medium results in RNA hyperadenylation. Strains were grown and analysed as described in Fig 1. Probes used were (A) 003, (B) 020, (C) 004, (D) 007, (E) 008, (F) 499, (G) 020, (H) 033, (I) 015, (J) 041, (K) 499 and (L) Phosphorlmager quantification of data from panel C.

Overexpression of Trf5p causes hyperadenylation. Trf5p was overexpressed in the absence of Trf4p and Rrp6p by growth of the GAL∷trf5, trf4Δ, rrp6Δ strain in galactose medium. (A,B) Polyadenylation of 5.8S+30 pre-ribosomal RNA. (C): Overexpression of TRF5 messenger RNA on galactose. Samples were processed as described in the legend to Fig 1. Probes used were (A) 020, (B) 499, (C) random primed probe to TRF5 open reading frame and (D) 403. (C,D) Poly(A)⁺ RNA only; PGK1 is an mRNA loading control.

The TRAMP5 complex shows Trf5p-dependent polyadenylation activity. (A) The predicted sequence of the carboxy-terminal region of Saccharomyces cerevisiae Trf5p was compared with the predicted open reading frames from the genomes of related yeasts. (B–D) In vitro poly(A) polymerase assays. (B) Comparison of the activity of purified Trf4p, Trf5p and the catalytically inactive Trf5p DADA mutant. (C) Comparison of polymerase activity with different nucleotide triphosphates. (D) Purified Trf5p retains poly(A) polymerase activity when purified from a strain lacking Trf4p. Proteins were isolated by means of C-terminal tandem affinity purification (TAP) tags from yeast grown to OD_{600 nm} 1.0 at 25°C in YPD. Trf4–TAP and Trf5–TAP concentrations were normalized by western blotting of the immunoprecipitated (IP) fractions using an anti-TAP antibody that recognizes the calmodulin binding peptide region of the tag. The substrate is a 5′-labelled 37-nt transcript derived from the pBS linker. Samples were taken at the indicated time points and resolved on a 12% polyacrylamide/urea gel. (E) SDS gel analysis of proteins co-precipitated with Trf5p. Marker, molecular weight markers; Trf5–TAP IP, precipitated proteins. Species identified by mass spectrometry are labelled. Rpl2Bp and Ccp1p (cytochrome c peroxidase) are abundant proteins that are likely to be contaminants.

Both Trf4p and Trf5p participate in the polyadenylation of ribosomal RNA precursors. Otherwise isogenic strains were grown at 25°C. Strains with GAL-regulated alleles were grown on 2% galactose and shifted to 2% glucose for 24 h before analysis. A 5 μg portion of total RNA and poly(A)⁺ RNA recovered from 50 μg total RNA was loaded per lane on 1.2% agarose/glyoxal (A–F) or 8% polyacrylamide/urea gels (G–M). (A–M) Northern hybridization with probes to pre-rRNA, rRNA and small nucleolar RNA species or the loading control TSA1, a highly expressed messenger RNA, the level of which is unaffected by exosome mutations. Probes used were (A) 003, (B) 020, (C) 004, (D) 007, (E) 008, (F) 499, (G) 020, (H) 015, (I) 041, (J) 499, (K) 202, (L) 214 and (M) 499. See supplementary Fig S1 online for descriptions of the pre-rRNA species and Supplementary Table S2 online for oligonucleotide probe sequences. RNA species marked with an asterisk are likely to represent as yet uncharacterized pre-rRNA degradation intermediates. (N) Graphs showing the fraction of each (pre-)rRNA species that is polyadenylated, calculated relative to the TSA1 mRNA control. The data are an average of three biological replicates; bars represent standard errors. Polyadenylated forms resolved on the polyacrylamide gels are indicated with pA. U24 is excised from the intron of the BEL1 pre-mRNA (Qu et al, 1995; Bousquet-Antonelli et al, 2000) and the band marked U24-int is likely to correspond to the species extended to the 5′ end of the intron (Qu et al, 1995; Bousquet-Antonelli et al, 2000).