SnoRNAs Are Synthesized from Two Classes of Polyadenylated Precursors
Transcriptional pulse stop of snR65 in GAL1::SNR65 (A) and GAL1::SNR65/rrp6Δ (B) strains. Transcription was induced for 60 min and then inhibited. I-pA and II-pA, polyadenylated precursors from respective termination sites; I^∗, oligoadenylated precursor from site I; M^∗, semimature species; M, mature snoRNA. (C) PhosphorImager quantification of data from (A) and (B) for mature snR65. Values are standardized to the U6 control and expressed relative to levels before induction.

Major Poly(A) Polymerase Pap1p Is Involved in Polyadenylation of snoRNA Precursors
(A) Northern analysis of polyadenylated pre-snoRNAs and 5S in rrp6Δ, rrp6Δ/pap1-2, and rrp6Δ/pap1-5 strains grown at 23°C or transferred to 37°C for total (lower panel, mature RNAs) and poly(A)⁺ fractions (upper panel).
(B) Western blot of Trf4-TAP in Trf4-TAP and Trf4-TAP/pap1-2 strains grown at 23°C or transferred to 37°C. Trf4-TAP and Mrf1 (loading control) detected by peroxidase-anti-peroxidase or protein-specific antibodies, respectively.
(C) SnR65 and snR13 in trf4Δ/pap1-2 and trf4Δ/pap1-5 strains grown at 23°C or shifted to 37°C.

Pap1 and Trf4 Are Essential for snoRNA Synthesis
Transcriptional pulse of snR65 in GAL1::SNR65 (A), GAL1::SNR65/pap1-5 (B), GAL1::SNR65/pap1-2 (C), and GAL1::SNR65/trf4Δ (D) strains. Transcription of snR65 was induced for 120–240 min as indicated. Temperature-sensitive pap1 cells were transferred to 37°C for 30 min before the pulse. RNA species are marked as in Figure 2. (E) PhosphorImager quantification of data from (A)–(C) for mature snR65. Values are standardized to the U6 control and expressed relative to levels before induction.

Model Correlating Termination, Polyadenylation, and Processing of snoRNAs
The majority of snoRNAs terminate at site I in a Nrd1/Nab3-dependent pathway; the mRNA cleavage and polyadenylation complex, denoted CP and marked as semitransparent, is inactive. Released pre-snoRNAs are adenylated by Trf4/5, further polyadenylated by Pap1, and rapidly processed in subsequent rounds of adenylation by Trf4/5 and exonucleolytic digestion by Rrp6. Transcripts that had passed site I are terminated by components of CP at site II, while the Nrd1/Nab3 complex, marked as semitransparent, is released or inactive. Site II precursors are polyadenylated exclusively by Pap1 and processed by the core exosome/Rrp6. The last few nucleotides are trimmed by Rrp6. Readthrough transcripts and defective snoRNPs are degraded by the exosome/Rrp6 in the course of processing.

Components of Nrd1/Nab3 and TRAMP Complexes Cooperate in Termination at Site I
(A) Readthrough transcription of the SNR13 gene in trf4Δ, nrd1-102, and GAL1::MTR4 mutants by primer extension from the downstream TRS31 gene. U1 is used as a control.
(B) Cotranscriptional association of Nrd1 with the SNR13 gene by ChIP in different strains carrying Nrd1-TAP. GAL1::MTR4 cells were pregrown on YPGal and transferred to YPD. Immunoprecipitated chromatin was amplified by qPCR using primers against the 3′ end of snR13 shown in the diagram below. ChIP signals were normalized by a background control from a nontranscribed region on chromosome V and corrected for Pol II occupancy along SNR13 using Pol II CTD-specific antibody (8WG16). Error bars reflect standard deviation of four experiments.

Polyadenylation and Processing of snoRNAs in TRAMP and Exosome Mutants
(A–D and F–H) Northern analysis of polyadenylated pre-snoRNAs in total and poly(A)⁺ fractions from various strains. U6 and SMX2 mRNA are loading controls for total and poly(A)⁺ RNAs, respectively. Site I species characteristic for rrp6Δ are indicated with an arrow in (B). Poly(A)⁺ fractions ([C], lanes 9 and 10, 19 and 20 and [H], lanes 19 and 20) or total RNA (D) deadenylated by RNase H treatment in the presence of oligo(dT). An arrow in (D) points at deadenylated pre-snR65 from terminator II; RNA size marker is on the right. To deplete Trf5 (C) or Mtr4 (F), trf4Δ/GAL1::TRF5 or GAL1::MTR4 cells were pregrown on YPGal and transferred to YPD; trf4Δ and nrd1-102 cells were grown at 23°C or transferred to 37°C (F); Dis3 was depleted by growth in the presence of doxycycline (H). (E) CR-RT-PCR of polyadenylated pre-snR65 terminated in region II in the trf4Δ strain. The number of added A residues is in subscript.

Polyadenylated snoRNA Species in the rrp6Δ Strain Originate from Transcription Terminators
(A, B, and G) SnoRNAs are oligoadenylated in the absence of Rrp6. Northern hybridization and CR-RT-PCR analysis of different box C/D and box H/ACA snoRNAs in wild-type and rrp6Δ strains (A and B). CR-RT-PCR for snR65 in the rrp6Δ/trf4Δ strain (G). Only the last few residues of mature ends are shown; undigested nucleotides and adenines are in bold. (C) Inactivation of Nop1 in a temperature-sensitive nop1-2 strain leads to accumulation of adenylated boxC/D snR13 and snR65 (marked with asterisks). (D) Northern hybridization of total and poly(A)⁺ RNA from the rrp6Δ strain for snR65 and snR13. U6 and SMX2 mRNA are loading controls for total and poly(A)⁺ RNAs, respectively. Two poly(A)⁺ populations are indicated by vertical lines. Arrows show major oligoadenylated precursor terminated at site I. Sequencing of RT-PCR products on polyadenylated precursors from the rrp6Δ strain is shown on the right. An arrowhead indicates the cleavage site in terminator I of snR13. (E) RNase H treatment of total RNA from the rrp6Δ strain in the presence of oligo(dT) for snR65. Arrows point at deadenylated species. RNA size marker is on the right. (F) CR-RT-PCR of polyadenylated pre-snR65 terminated in region II in the rrp6Δ strain. The number of added A residues is in subscript, and numerals in parentheses denote number of clones.