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Polyadenylation linked to transcription termination directs the processing of snoRNA precursors in yeast.

Grzechnik P, Kufel J - Mol. Cell (2008)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Genetics and Biotechnology, Faculty of Biology, University of Warsaw, Pawinskiego 5a, 02-106 Warsaw, Poland.

ABSTRACT
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.

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Pap1 and Trf4 Are Essential for snoRNA SynthesisTranscriptional 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.
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fig6: Pap1 and Trf4 Are Essential for snoRNA SynthesisTranscriptional 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.

Mentions: From the data presented so far, it appears that polyadenylation of pre-snoRNAs is related to termination. The most relevant question is whether it is the normal or secondary pathway of their synthesis. To address this, a transcriptional pulse of the inducible GAL1::SNR65 was carried out in wild-type, pap1-2, pap1-5, and trf4Δ strains (Figure 6). SnR65 synthesis in wild-type and pap1-5 cells at the permissive temperature proceeds as already described (see Figure 2A), via accumulation of polyadenylated II-pA and oligoadenylated I∗ precursors as well as Rrp6-dependent M∗ intermediates, followed by the buildup of the mature RNA. After transfer of pap1-5 cells to 37°C for 30 min prior to the pulse, precursors derived from both terminators are markedly reduced and accumulation of mature species significantly inhibited (Figure 6E). The pulse in the pap1-2 strain at both temperatures was very weak, yielding little, if any, polyadenylated precursors and no increase in mature snR65 (Figures 6C and 6E). The effects in pap1 mutants are analogous to induction of hypoadenylated mRNAs (Hilleren et al., 2001; Milligan et al., 2005) and are not due to the inhibition of Pol II, as transcription is not affected (Birse et al., 1998; Ciais et al., 2008). These facts point to a direct role of Pap1 in the synthesis of snoRNAs. In the trf4Δ strain, II-pA species readily accumulate, but I∗ and M∗ are totally absent, which confirms that they derive from I-pA and not II-pA precursors. Despite the presence of polyadenylated precursors, mature snR65 is not produced efficiently: only after 4 hr of the pulse did the snoRNA level show a moderate increase. As transcription rates are similar in trf4Δ and wild-type cells (Figure S7), this outcome probably results from a slower, ineffective processing. The appearance of a characteristic ladder of nonpolyadenylated intermediates in the absence of Trf4 indicates that it may be involved in the synthesis of mature snoRNAs via rounds of adenylation followed by exonucleolytic trimming.


Polyadenylation linked to transcription termination directs the processing of snoRNA precursors in yeast.

Grzechnik P, Kufel J - Mol. Cell (2008)

Pap1 and Trf4 Are Essential for snoRNA SynthesisTranscriptional 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.
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fig6: Pap1 and Trf4 Are Essential for snoRNA SynthesisTranscriptional 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.
Mentions: From the data presented so far, it appears that polyadenylation of pre-snoRNAs is related to termination. The most relevant question is whether it is the normal or secondary pathway of their synthesis. To address this, a transcriptional pulse of the inducible GAL1::SNR65 was carried out in wild-type, pap1-2, pap1-5, and trf4Δ strains (Figure 6). SnR65 synthesis in wild-type and pap1-5 cells at the permissive temperature proceeds as already described (see Figure 2A), via accumulation of polyadenylated II-pA and oligoadenylated I∗ precursors as well as Rrp6-dependent M∗ intermediates, followed by the buildup of the mature RNA. After transfer of pap1-5 cells to 37°C for 30 min prior to the pulse, precursors derived from both terminators are markedly reduced and accumulation of mature species significantly inhibited (Figure 6E). The pulse in the pap1-2 strain at both temperatures was very weak, yielding little, if any, polyadenylated precursors and no increase in mature snR65 (Figures 6C and 6E). The effects in pap1 mutants are analogous to induction of hypoadenylated mRNAs (Hilleren et al., 2001; Milligan et al., 2005) and are not due to the inhibition of Pol II, as transcription is not affected (Birse et al., 1998; Ciais et al., 2008). These facts point to a direct role of Pap1 in the synthesis of snoRNAs. In the trf4Δ strain, II-pA species readily accumulate, but I∗ and M∗ are totally absent, which confirms that they derive from I-pA and not II-pA precursors. Despite the presence of polyadenylated precursors, mature snR65 is not produced efficiently: only after 4 hr of the pulse did the snoRNA level show a moderate increase. As transcription rates are similar in trf4Δ and wild-type cells (Figure S7), this outcome probably results from a slower, ineffective processing. The appearance of a characteristic ladder of nonpolyadenylated intermediates in the absence of Trf4 indicates that it may be involved in the synthesis of mature snoRNAs via rounds of adenylation followed by exonucleolytic trimming.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Genetics and Biotechnology, Faculty of Biology, University of Warsaw, Pawinskiego 5a, 02-106 Warsaw, Poland.

ABSTRACT
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.

Show MeSH