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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-tRNAs are targets for the nuclear RNA surveillance machinery. (A) Average densities of reads mapped to intron-containing tRNAs. tRNA exons and introns have various lengths; all exons and introns were divided into three bins and density of reads in each bin is displayed. (B, C) Average densities of reads of all tRNAs associated with the indicated proteins are plotted with respect to start (B) and end of the tRNA (C). (D, E) Alignments of high-throughput sequencing reads of RNAs associated with the indicated proteins to 3′ extended pre-tRNAs (D) and 5′ extended pre-tRNAs (E). Grey boxes mark mature tRNA sequences and numbering indicates nucleotide positions with respect to nucleotide +1 of the tRNA. Mismatches and deletions in sequencing reads are displayed in red. Nrd1 and Nab3 consensus-binding motifs are underlined. Numbers in brackets indicate the frequency with which each specific sequence was recovered in reads per million mapped sequences. (F, G) Northern analyses of total RNA from BY4741, GAL∷nrd1CIDΔ, GAL∷nrd1 and GAL∷nab3 strains. Oligonucleotide probes are given with the probe number in brackets. A schematic representation of the identified species is shown. (H) Quantification of pre-tRNA relative to mature tRNA is shown; expression at 12 versus 0 h is set to 1 for the WT. Average of three biological replicates is shown with s.d. See also Supplementary Figure S6.
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f7: Pre-tRNAs are targets for the nuclear RNA surveillance machinery. (A) Average densities of reads mapped to intron-containing tRNAs. tRNA exons and introns have various lengths; all exons and introns were divided into three bins and density of reads in each bin is displayed. (B, C) Average densities of reads of all tRNAs associated with the indicated proteins are plotted with respect to start (B) and end of the tRNA (C). (D, E) Alignments of high-throughput sequencing reads of RNAs associated with the indicated proteins to 3′ extended pre-tRNAs (D) and 5′ extended pre-tRNAs (E). Grey boxes mark mature tRNA sequences and numbering indicates nucleotide positions with respect to nucleotide +1 of the tRNA. Mismatches and deletions in sequencing reads are displayed in red. Nrd1 and Nab3 consensus-binding motifs are underlined. Numbers in brackets indicate the frequency with which each specific sequence was recovered in reads per million mapped sequences. (F, G) Northern analyses of total RNA from BY4741, GAL∷nrd1CIDΔ, GAL∷nrd1 and GAL∷nab3 strains. Oligonucleotide probes are given with the probe number in brackets. A schematic representation of the identified species is shown. (H) Quantification of pre-tRNA relative to mature tRNA is shown; expression at 12 versus 0 h is set to 1 for the WT. Average of three biological replicates is shown with s.d. See also Supplementary Figure S6.

Mentions: Pre-tRNAs are transcribed with a 5′ leader and 3′ trailer and in some cases also contain introns. These are removed during tRNA maturation, while the 3′ CCA tail is added and many base modifications are introduced. Nrd1, Nab3 and Trf4 were associated with many pre-tRNA fragments, which generally contained introns (Figure 7A), 5′ leaders (Figure 7B) or 3′ extensions (Figure 7C). Almost no tRNA recovered carried the 3′ CCA tail, whereas many retained the 3′ oligo(U) Pol III termination signal, followed by a non-encoded oligo(A) tail, or just an oligo(A) tail following the coding region (Figure 7D and data not shown). The recovery of many oligoadenylated RNAs that extend to the Pol III terminator indicated that tRNA-like species detected in CRAC analyses are derived from bona fide pre-tRNAs. In the case of tRNAIle(AAU), which is reported to be edited (Auxilien et al, 1996), the Nrd1 CRAC recovered only RNAs that had failed to undergo editing of the anticodon loop (data not shown). We conclude that the RNAs recovered were predominately derived from pre-tRNA species rather than mature tRNAs. Pre-tRNAs recovered with Nrd1–Nab3 generally carried consensus-binding motifs and mutations were frequently found within and around the GUAA/G and UCUU/CUUG sequences (underlined in Figure 7D and E), indicating direct protein binding.


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

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

Pre-tRNAs are targets for the nuclear RNA surveillance machinery. (A) Average densities of reads mapped to intron-containing tRNAs. tRNA exons and introns have various lengths; all exons and introns were divided into three bins and density of reads in each bin is displayed. (B, C) Average densities of reads of all tRNAs associated with the indicated proteins are plotted with respect to start (B) and end of the tRNA (C). (D, E) Alignments of high-throughput sequencing reads of RNAs associated with the indicated proteins to 3′ extended pre-tRNAs (D) and 5′ extended pre-tRNAs (E). Grey boxes mark mature tRNA sequences and numbering indicates nucleotide positions with respect to nucleotide +1 of the tRNA. Mismatches and deletions in sequencing reads are displayed in red. Nrd1 and Nab3 consensus-binding motifs are underlined. Numbers in brackets indicate the frequency with which each specific sequence was recovered in reads per million mapped sequences. (F, G) Northern analyses of total RNA from BY4741, GAL∷nrd1CIDΔ, GAL∷nrd1 and GAL∷nab3 strains. Oligonucleotide probes are given with the probe number in brackets. A schematic representation of the identified species is shown. (H) Quantification of pre-tRNA relative to mature tRNA is shown; expression at 12 versus 0 h is set to 1 for the WT. Average of three biological replicates is shown with s.d. See also Supplementary Figure S6.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f7: Pre-tRNAs are targets for the nuclear RNA surveillance machinery. (A) Average densities of reads mapped to intron-containing tRNAs. tRNA exons and introns have various lengths; all exons and introns were divided into three bins and density of reads in each bin is displayed. (B, C) Average densities of reads of all tRNAs associated with the indicated proteins are plotted with respect to start (B) and end of the tRNA (C). (D, E) Alignments of high-throughput sequencing reads of RNAs associated with the indicated proteins to 3′ extended pre-tRNAs (D) and 5′ extended pre-tRNAs (E). Grey boxes mark mature tRNA sequences and numbering indicates nucleotide positions with respect to nucleotide +1 of the tRNA. Mismatches and deletions in sequencing reads are displayed in red. Nrd1 and Nab3 consensus-binding motifs are underlined. Numbers in brackets indicate the frequency with which each specific sequence was recovered in reads per million mapped sequences. (F, G) Northern analyses of total RNA from BY4741, GAL∷nrd1CIDΔ, GAL∷nrd1 and GAL∷nab3 strains. Oligonucleotide probes are given with the probe number in brackets. A schematic representation of the identified species is shown. (H) Quantification of pre-tRNA relative to mature tRNA is shown; expression at 12 versus 0 h is set to 1 for the WT. Average of three biological replicates is shown with s.d. See also Supplementary Figure S6.
Mentions: Pre-tRNAs are transcribed with a 5′ leader and 3′ trailer and in some cases also contain introns. These are removed during tRNA maturation, while the 3′ CCA tail is added and many base modifications are introduced. Nrd1, Nab3 and Trf4 were associated with many pre-tRNA fragments, which generally contained introns (Figure 7A), 5′ leaders (Figure 7B) or 3′ extensions (Figure 7C). Almost no tRNA recovered carried the 3′ CCA tail, whereas many retained the 3′ oligo(U) Pol III termination signal, followed by a non-encoded oligo(A) tail, or just an oligo(A) tail following the coding region (Figure 7D and data not shown). The recovery of many oligoadenylated RNAs that extend to the Pol III terminator indicated that tRNA-like species detected in CRAC analyses are derived from bona fide pre-tRNAs. In the case of tRNAIle(AAU), which is reported to be edited (Auxilien et al, 1996), the Nrd1 CRAC recovered only RNAs that had failed to undergo editing of the anticodon loop (data not shown). We conclude that the RNAs recovered were predominately derived from pre-tRNA species rather than mature tRNAs. Pre-tRNAs recovered with Nrd1–Nab3 generally carried consensus-binding motifs and mutations were frequently found within and around the GUAA/G and UCUU/CUUG sequences (underlined in Figure 7D and E), indicating direct protein binding.

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