Limits...
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.

Show MeSH

Related in: MedlinePlus

Crosslinking experiments recover known targets for the nuclear RNA surveillance machinery. Densities in hits per million of high-throughput sequencing reads of the indicated IP mapped to SNR13 (A), SNR3 (B), NRD1 (C), CTH2 (D), IGS1-R CUT (E) and SRG1-SER3 (F). Open reading frames are represented by arrows, ncRNAs by boxes. UTRs and terminator elements are indicated where appropriate. (E) 5S and 25S transcripts are represented by bold arrows and cryptic ncRNAs by slim arrows. See also Supplementary Figure S3.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3102002&req=5

f3: Crosslinking experiments recover known targets for the nuclear RNA surveillance machinery. Densities in hits per million of high-throughput sequencing reads of the indicated IP mapped to SNR13 (A), SNR3 (B), NRD1 (C), CTH2 (D), IGS1-R CUT (E) and SRG1-SER3 (F). Open reading frames are represented by arrows, ncRNAs by boxes. UTRs and terminator elements are indicated where appropriate. (E) 5S and 25S transcripts are represented by bold arrows and cryptic ncRNAs by slim arrows. See also Supplementary Figure S3.

Mentions: Previous analyses of SNR13 identified two terminator elements downstream of the 3′ end of the snoRNA (Steinmetz et al, 2001; Carroll et al, 2004). These include consensus Nrd1 and Nab3 binding sequences and their mutation leads to transcriptional read-through (Carroll et al, 2004). Pre-snR13 was bound by Nrd1 and Nab3, but also interacted with Trf4. The majority of reads were mapped to the downstream terminator elements, rather than the mature snoRNA (Figures 1E and 3A; Supplementary Figure S3). Nop58 crosslinked to snR13 and many other boxC/D snoRNAs, but preferentially associated with the internal boxD′ element, which is different from the preferred surveillance factor binding sites (Figure 1E). Terminator I was bound by both Nrd1 and Nab3, with the reads covering the consensus-binding sequences (Figure 3A; Supplementary Figure S3).


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

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

Crosslinking experiments recover known targets for the nuclear RNA surveillance machinery. Densities in hits per million of high-throughput sequencing reads of the indicated IP mapped to SNR13 (A), SNR3 (B), NRD1 (C), CTH2 (D), IGS1-R CUT (E) and SRG1-SER3 (F). Open reading frames are represented by arrows, ncRNAs by boxes. UTRs and terminator elements are indicated where appropriate. (E) 5S and 25S transcripts are represented by bold arrows and cryptic ncRNAs by slim arrows. See also Supplementary Figure S3.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Crosslinking experiments recover known targets for the nuclear RNA surveillance machinery. Densities in hits per million of high-throughput sequencing reads of the indicated IP mapped to SNR13 (A), SNR3 (B), NRD1 (C), CTH2 (D), IGS1-R CUT (E) and SRG1-SER3 (F). Open reading frames are represented by arrows, ncRNAs by boxes. UTRs and terminator elements are indicated where appropriate. (E) 5S and 25S transcripts are represented by bold arrows and cryptic ncRNAs by slim arrows. See also Supplementary Figure S3.
Mentions: Previous analyses of SNR13 identified two terminator elements downstream of the 3′ end of the snoRNA (Steinmetz et al, 2001; Carroll et al, 2004). These include consensus Nrd1 and Nab3 binding sequences and their mutation leads to transcriptional read-through (Carroll et al, 2004). Pre-snR13 was bound by Nrd1 and Nab3, but also interacted with Trf4. The majority of reads were mapped to the downstream terminator elements, rather than the mature snoRNA (Figures 1E and 3A; Supplementary Figure S3). Nop58 crosslinked to snR13 and many other boxC/D snoRNAs, but preferentially associated with the internal boxD′ element, which is different from the preferred surveillance factor binding sites (Figure 1E). Terminator I was bound by both Nrd1 and Nab3, with the reads covering the consensus-binding sequences (Figure 3A; Supplementary Figure S3).

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