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The nuclear RNA polymerase II surveillance system targets polymerase III transcripts.

Wlotzka W, Kudla G, Granneman S, Tollervey D - EMBO J. (2011)

Bottom Line: Mapping of micro-deletions and substitutions allowed clear definition of preferred, in vivo Nab3 and Nrd1 binding sites.Surveillance targets were enriched for non-encoded A-rich tails.These were generally very short (1–5 nt), potentially explaining why adenylation destabilizes these RNAs while stabilizing mRNAs with long poly(A) tails.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, UK.

ABSTRACT
A key question in nuclear RNA surveillance is how target RNAs are recognized. To address this, we identified in vivo binding sites for nuclear RNA surveillance factors, Nrd1, Nab3 and the Trf4/5–Air1/2–Mtr4 polyadenylation (TRAMP) complex poly(A) polymerase Trf4, by UV crosslinking. Hit clusters were reproducibly found over known binding sites on small nucleolar RNAs (snoRNAs), pre-mRNAs and cryptic, unstable non-protein-coding RNAs (ncRNAs) ('CUTs'), along with ~642 predicted long anti-sense ncRNAs (asRNAs), ~178 intergenic ncRNAs and, surprisingly, ~1384 mRNAs. Five putative asRNAs tested were confirmed to exist and were stabilized by loss of Nrd1, Nab3 or Trf4. Mapping of micro-deletions and substitutions allowed clear definition of preferred, in vivo Nab3 and Nrd1 binding sites. Nrd1 and Nab3 were believed to be Pol II specific but, unexpectedly, bound many oligoadenylated Pol III transcripts, predominately pre-tRNAs. Depletion of Nrd1 or Nab3 stabilized tested Pol III transcripts and their oligoadenylation was dependent on Nrd1–Nab3 and TRAMP. Surveillance targets were enriched for non-encoded A-rich tails. These were generally very short (1–5 nt), potentially explaining why adenylation destabilizes these RNAs while stabilizing mRNAs with long poly(A) tails.

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Pre-Rrp1 is polyadenylated and targeted by the surveillance machinery. (A) Densities of high-throughput sequencing reads mapped to pre-RPR1. (B) Two-dimensional structure of RPR1 according to Srisawat et al (2002). High-throughput sequencing reads mapped to RPR1 are highlighted. Trf4 crosslinks overlapped with Nab3 hits and have been omitted for clarity. (C) Alignment of high-throughput sequencing reads of RNAs in the indicated IP to pre-RPR1. Grey boxes mark the mature RPR1 sequence and numbering indicates the nucleotide position with respect to nucleotide +1 in the RPR1 gene. Mismatches and deletions in sequencing reads are displayed in red. Numbers in brackets indicate the frequency with which each specific sequence was recovered in reads per million mapped sequences. (D) Northern analyses of total and poly(A)+ RNA from GAL∷nrd1, GAL∷nab3 and BY4741 strains. Quantification of expression levels of polyadenylated pre-RPR1 relative to TSA1 mRNA is displayed. Ratio of expression after 12 h compared with 0 h is set to 1 for WT and given as average of three biological replicates with s.d.
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f6: Pre-Rrp1 is polyadenylated and targeted by the surveillance machinery. (A) Densities of high-throughput sequencing reads mapped to pre-RPR1. (B) Two-dimensional structure of RPR1 according to Srisawat et al (2002). High-throughput sequencing reads mapped to RPR1 are highlighted. Trf4 crosslinks overlapped with Nab3 hits and have been omitted for clarity. (C) Alignment of high-throughput sequencing reads of RNAs in the indicated IP to pre-RPR1. Grey boxes mark the mature RPR1 sequence and numbering indicates the nucleotide position with respect to nucleotide +1 in the RPR1 gene. Mismatches and deletions in sequencing reads are displayed in red. Numbers in brackets indicate the frequency with which each specific sequence was recovered in reads per million mapped sequences. (D) Northern analyses of total and poly(A)+ RNA from GAL∷nrd1, GAL∷nab3 and BY4741 strains. Quantification of expression levels of polyadenylated pre-RPR1 relative to TSA1 mRNA is displayed. Ratio of expression after 12 h compared with 0 h is set to 1 for WT and given as average of three biological replicates with s.d.

Mentions: RPR1 encodes the RNA component of RNase P and is transcribed by Pol III as a precursor containing a 5′ leader and 3′ trailer, removal of which requires RNP assembly (see Srisawat et al (2002) and references therein). CRAC revealed association of Nab3 and Trf4 with the 5′ leader and Nrd1 and Trf4 with the 3′ trailer of pre-RPR1 (Figure 6A–C). Sequences recovered with Nrd1 and Trf4 did not contain the full 3′ trailer up to the transcription stop site but carried extensions with non-encoded oligo(A) tails (Figure 6C). Trf4 and Nab3 bound an overlapping set of sites within the mature RNA, which are brought into proximity in the predicted secondary structure (Figure 6B).


The nuclear RNA polymerase II surveillance system targets polymerase III transcripts.

Wlotzka W, Kudla G, Granneman S, Tollervey D - EMBO J. (2011)

Pre-Rrp1 is polyadenylated and targeted by the surveillance machinery. (A) Densities of high-throughput sequencing reads mapped to pre-RPR1. (B) Two-dimensional structure of RPR1 according to Srisawat et al (2002). High-throughput sequencing reads mapped to RPR1 are highlighted. Trf4 crosslinks overlapped with Nab3 hits and have been omitted for clarity. (C) Alignment of high-throughput sequencing reads of RNAs in the indicated IP to pre-RPR1. Grey boxes mark the mature RPR1 sequence and numbering indicates the nucleotide position with respect to nucleotide +1 in the RPR1 gene. Mismatches and deletions in sequencing reads are displayed in red. Numbers in brackets indicate the frequency with which each specific sequence was recovered in reads per million mapped sequences. (D) Northern analyses of total and poly(A)+ RNA from GAL∷nrd1, GAL∷nab3 and BY4741 strains. Quantification of expression levels of polyadenylated pre-RPR1 relative to TSA1 mRNA is displayed. Ratio of expression after 12 h compared with 0 h is set to 1 for WT and given as average of three biological replicates with s.d.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3102002&req=5

f6: Pre-Rrp1 is polyadenylated and targeted by the surveillance machinery. (A) Densities of high-throughput sequencing reads mapped to pre-RPR1. (B) Two-dimensional structure of RPR1 according to Srisawat et al (2002). High-throughput sequencing reads mapped to RPR1 are highlighted. Trf4 crosslinks overlapped with Nab3 hits and have been omitted for clarity. (C) Alignment of high-throughput sequencing reads of RNAs in the indicated IP to pre-RPR1. Grey boxes mark the mature RPR1 sequence and numbering indicates the nucleotide position with respect to nucleotide +1 in the RPR1 gene. Mismatches and deletions in sequencing reads are displayed in red. Numbers in brackets indicate the frequency with which each specific sequence was recovered in reads per million mapped sequences. (D) Northern analyses of total and poly(A)+ RNA from GAL∷nrd1, GAL∷nab3 and BY4741 strains. Quantification of expression levels of polyadenylated pre-RPR1 relative to TSA1 mRNA is displayed. Ratio of expression after 12 h compared with 0 h is set to 1 for WT and given as average of three biological replicates with s.d.
Mentions: RPR1 encodes the RNA component of RNase P and is transcribed by Pol III as a precursor containing a 5′ leader and 3′ trailer, removal of which requires RNP assembly (see Srisawat et al (2002) and references therein). CRAC revealed association of Nab3 and Trf4 with the 5′ leader and Nrd1 and Trf4 with the 3′ trailer of pre-RPR1 (Figure 6A–C). Sequences recovered with Nrd1 and Trf4 did not contain the full 3′ trailer up to the transcription stop site but carried extensions with non-encoded oligo(A) tails (Figure 6C). Trf4 and Nab3 bound an overlapping set of sites within the mature RNA, which are brought into proximity in the predicted secondary structure (Figure 6B).

Bottom Line: Mapping of micro-deletions and substitutions allowed clear definition of preferred, in vivo Nab3 and Nrd1 binding sites.Surveillance targets were enriched for non-encoded A-rich tails.These were generally very short (1–5 nt), potentially explaining why adenylation destabilizes these RNAs while stabilizing mRNAs with long poly(A) tails.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, UK.

ABSTRACT
A key question in nuclear RNA surveillance is how target RNAs are recognized. To address this, we identified in vivo binding sites for nuclear RNA surveillance factors, Nrd1, Nab3 and the Trf4/5–Air1/2–Mtr4 polyadenylation (TRAMP) complex poly(A) polymerase Trf4, by UV crosslinking. Hit clusters were reproducibly found over known binding sites on small nucleolar RNAs (snoRNAs), pre-mRNAs and cryptic, unstable non-protein-coding RNAs (ncRNAs) ('CUTs'), along with ~642 predicted long anti-sense ncRNAs (asRNAs), ~178 intergenic ncRNAs and, surprisingly, ~1384 mRNAs. Five putative asRNAs tested were confirmed to exist and were stabilized by loss of Nrd1, Nab3 or Trf4. Mapping of micro-deletions and substitutions allowed clear definition of preferred, in vivo Nab3 and Nrd1 binding sites. Nrd1 and Nab3 were believed to be Pol II specific but, unexpectedly, bound many oligoadenylated Pol III transcripts, predominately pre-tRNAs. Depletion of Nrd1 or Nab3 stabilized tested Pol III transcripts and their oligoadenylation was dependent on Nrd1–Nab3 and TRAMP. Surveillance targets were enriched for non-encoded A-rich tails. These were generally very short (1–5 nt), potentially explaining why adenylation destabilizes these RNAs while stabilizing mRNAs with long poly(A) tails.

Show MeSH
Related in: MedlinePlus