Polyadenylation Linked to Transcription Termination Directs the Processing of snoRNA Precursors in Yeast

Summary Transcription termination by RNA polymerase II is coupled to transcript 3′ end formation. A large cleavage and polyadenylation complex containing the major poly(A) polymerase Pap1 produces mRNA 3′ ends, whereas those of nonpolyadenylated snoRNAs in yeast are formed either by endonucleolytic cleavage or by termination, followed by trimming by the nuclear exosome. We show that synthesis of independently transcribed snoRNAs involves default polyadenylation of two classes of precursors derived from termination at a main Nrd1/Nab3-dependent site or a “fail-safe” mRNA-like signal. Poly(A) tails are added by Pap1 to both forms, whereas the alternative poly(A) polymerase Tfr4 adenylates major precursors and processing intermediates to facilitate further polyadenylation by Pap1 and maturation by the exosome/Rrp6. A more important role of Trf4/TRAMP, however, is to enhance Nrd1 association with snoRNA genes. We propose a model in which polyadenylation of pre-snoRNAs is a key event linking their transcription termination, 3′ end processing, and degradation.


General RNA methods
Total RNA from yeast cells was isolated using a hot phenol procedure (Schmitt et al., 1990).
Northern hybridization and primer extension were essentially as described (Tollervey and Mattaj, 1987). Radioactive probes were either 5'-end γ-32 P-labelled oligoprobes or α-32 P internally labelled random-primed probe (for SmX2 mRNA) prepared using PCR product as template and DECAprime II Kit (Ambion). 8μg of total RNA or 0.5μg of poly(A) + RNA and 2μg of total RNA in the case of purified poly(A) + samples were separated on 6% denaturing polyacrylamide-urea gels, transferred onto nylon membranes and hybridized with oligonucleotide probes listed in supplementary Table S2.

Western blot analysis
Western blot analysis was performed using peroxidase-anti-peroxidase antibody to detect Nrd1-TAP and Trf4-TAP and polyclonal anti-Mrf1 antibody followed by horseradish peroxidase-conjugated goat anti-rabbit antibody.

Calculation of ChIP values
Nrd1-TAP occupancy at SNR13 in trf4Δ, trf4-236 and GAL1::MTR4 (in nonpermissive conditions, growth in glucose) in Figure 5B was compared to the level in the otherwise wildtype Nrd1-TAP control or Nrd1-TAP/GAL1::MTR4 cells (in permissive conditions, growth on galactose).
ChIP values for Nrd1-TAP were quantified using the formula 2 -ΔΔCt = 2 -((Ct IP target gene -Ct Input target gene) -(Ct IP control -Ct Input control)) , where "Ct IP" and "Ct Input target gene" are cycle numbers for the SNR13 gene and "Ct IP" and "Ct Input control" are cycle numbers for non-coding region on chromosome V. ChIP values for Pol II were determined using 2 -ΔCt = 2 -(ct IP -ct background) , where "Ct IP" is cycle number for immunoprecipitate and "Ct background" is cycle number for control without antibodies. ChIP levels for Nrd1-TAP in different strains were corrected for Pol II occupancy. rrp6Δ/pap1-2 as pap1-2 but RRP6:: K.lactis URA3 (Milligan et al., 2005) rrp6Δ/pap1-5 as pap1-5 but RRP6:: K.lactis URA3 (Milligan et al., 2005)     and SMX2 mRNA (lane 2) are used as loading controls for total and poly(A) + RNAs, respectively.

Figure S2
Both I-pA and II-pA precursors are detected during the transcriptional pulse in wild-type cells.
RT-PCR analysis of polyadenylated pre-snR65 in GAL1::SNR65 strain where transcription was induced by addition of galactose for times indicated. Reverse transcription was performed using using ADAPT-oligo(dT) 30 and cDNA was amplified using ADAPToligo(dT) 30 and a primer specific for mature snR65. The asterisk indicates primer-dimers.

Figure S3
Transcriptional pulse of snR65 under the control of the GAL1 promoter generates mature (M) snoRNA in wild-type, trf4Δ, pap1-2 strains but only untrimmed semi-mature (M*) species in rrp6Δ cells. Cells were grown at 23˚C in SC medium (2% raffinose, 0.08% glucose) and transcription of snR65 was induced for times indicated by addition of galactose. I-pA and II-pA, polyadenylated precursors from respective termination sites; I*, oligoadenylated precursor from site I; M*, semi-mature species; M, mature snoRNA.

Figure S4
Deadenylases are not involved in removal of poly(A) tails.
Northern analysis of snR65 in ccr4Δ/caf1Δ and ccr4Δ/pan2Δ strains. Total RNA (lower panel with mature snoRNA) and the poly(A) + fraction (upper panel). U6 and SmX2 mRNA, loading controls for total and poly(A) + RNAs, respectively.

Figure S5
TRAMP components Air1/2 are also involved in snoRNA processing. Northern analysis of polyadenylated snR65 in trf4Δ, air1Δ, air2Δ, air1Δ/air2Δ and trf4-ts836/trf5Δ strains. Only deletion of both Air proteins result in the phenotype comparable to that in the trf4Δ strain as 11 had been observed for several effects characteristic for TRAMP mutants (LaCava et al., 2005;Wyers et al., 2005). U6 is used as a control.

Figure S6
Mutation in Nrd1 and lack of Trf4 shift polyadenylated snR65 precursors towards site II.
RT-PCR analysis of polyadenylated pre-snR65 in wild-type, nrd1-102 and trf4Δ strains grown at 23˚C or shifted to 37˚C for 2 hours. Reverse transcription was performed using using ADAPT-oligo(dT) 30 and the cDNA was amplified using ADAPT-oligo(dT) 30 and a primer specific for mature snR65.

Figure S7
Transcription rate of endogenous snR13 is not altered in the absence of Trf4 and by the trf4-236 mutation (A) or following depletion of Mtr4 (B). SnR65 under the control of GAL1 promoter is transcribed with similar rates in wild-type and trf4Δ strains (C).
Pol II occupancy along SNR13 or SNR65 was analysed by ChIP using 8WG16 antibodies against the CTD of Pol II in Nrd1-TAP (wt), Nrd1-TAP/trf4Δ (trf4Δ)

Figure S8
Deletion of Trf4 and depletion of Mtr4 do not affect the level of Nrd1 protein.