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The Sm complex is required for the processing of non-coding RNAs by the exosome.

Coy S, Volanakis A, Shah S, Vasiljeva L - PLoS ONE (2013)

Bottom Line: Additionally, we demonstrate that the same pathway is involved in the maturation of snRNAs.We also show that telomerase RNA accumulates in Schizosaccharomyces pombe exosome mutants, suggesting a conserved role for the exosome in processing and degradation of telomerase RNA.In summary, our data provide important mechanistic insight into the regulation of exosome dependent RNA processing as well as telomerase RNA biogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Oxford, Oxford, United Kingdom.

ABSTRACT
A key question in the field of RNA regulation is how some exosome substrates, such as spliceosomal snRNAs and telomerase RNA, evade degradation and are processed into stable, functional RNA molecules. Typical feature of these non-coding RNAs is presence of the Sm complex at the 3'end of the mature RNA molecule. Here, we report that in Saccharomyces cerevisiae presence of intact Sm binding site is required for the exosome-mediated processing of telomerase RNA from a polyadenylated precursor into its mature form and is essential for its function in elongating telomeres. Additionally, we demonstrate that the same pathway is involved in the maturation of snRNAs. Furthermore, the insertion of an Sm binding site into an unstable RNA that is normally completely destroyed by the exosome, leads to its partial stabilization. We also show that telomerase RNA accumulates in Schizosaccharomyces pombe exosome mutants, suggesting a conserved role for the exosome in processing and degradation of telomerase RNA. In summary, our data provide important mechanistic insight into the regulation of exosome dependent RNA processing as well as telomerase RNA biogenesis.

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Exosome mutants accumulate poly(A)+ telomerase RNA.A) Schematic diagram describing the positions of TLC1 (S. cerevisiae) and TER1 (S. pombe) probes for telomerase RNA. Probes are depicted as black bars. B) Accumulation of poly(A)+ TLC1 RNA in S. cerevisiae exosome mutant strains. Northern blot analysis of total RNA from WT (YF336), lane 1; rrp47Δ (YF1465), lane 2; WT (YF1444), lane 3 and rrp6Δ (YSB2244), lane 4. RNA was resolved on a 5% PAGE and TLC1 RNA was visualized with the probe depicted in (A). Positions of poly(A)+ and poly(A)− TLC1 RNAs are indicated with grey boxes. Methylene-Blue stained 18 and 25S rRNAs are shown below and serve as a loading control. C) Exonucleolytic activity of the exosome is required for the processing of poly(A)+ TLC1 RNA. Northern blot analysis of total RNA, lanes 1, 3–5 and 7–9 or oligo-dT purified RNA, lanes 2 and 6. RNA was isolated from WT (YF336), lanes 1 and 2; rrp47Δ (YF1465), lane 3; WT (YF1444), lane 4; rrp6Δ (YSB2244), lanes 5 and 6; WT (YF1977), lane 7; dis3 (D171A) (YF1978), lane 8 and dis3 (D551N) (YF1979), lane 9. RNA was resolved on a 1.2% agarose gel and probed for TLC1 RNA. 25 and 18S rRNAs are shown below. Numbers below indicate fold increase in poly(A)+, poly(A) − TLC1 RNA levels relative to WT and poly(A)+/poly(A) − ratio. D) Accumulation of poly(A)+ TER1 RNA in S. pombe exosome mutant strains. Northern blot analysis of total RNA, lanes 1, 3 and 5, or oligo-dT purified RNA, lanes 2, 4 and 6 from WT (YP34), lanes 1 and 2; rrp6Δ (YP35), lanes 3 and 4; dis3-54 (YP50), lanes 5 and 6. RNA was resolved on a 5% PAGE and TER1 RNA was visualized with the probe depicted in (A). Positions of poly(A)+ and poly(A) − TER1 RNAs are indicated with grey boxes. 18S rRNA is shown below. Numbers below indicate fold increase in RNA levels relative to WT.
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pone-0065606-g001: Exosome mutants accumulate poly(A)+ telomerase RNA.A) Schematic diagram describing the positions of TLC1 (S. cerevisiae) and TER1 (S. pombe) probes for telomerase RNA. Probes are depicted as black bars. B) Accumulation of poly(A)+ TLC1 RNA in S. cerevisiae exosome mutant strains. Northern blot analysis of total RNA from WT (YF336), lane 1; rrp47Δ (YF1465), lane 2; WT (YF1444), lane 3 and rrp6Δ (YSB2244), lane 4. RNA was resolved on a 5% PAGE and TLC1 RNA was visualized with the probe depicted in (A). Positions of poly(A)+ and poly(A)− TLC1 RNAs are indicated with grey boxes. Methylene-Blue stained 18 and 25S rRNAs are shown below and serve as a loading control. C) Exonucleolytic activity of the exosome is required for the processing of poly(A)+ TLC1 RNA. Northern blot analysis of total RNA, lanes 1, 3–5 and 7–9 or oligo-dT purified RNA, lanes 2 and 6. RNA was isolated from WT (YF336), lanes 1 and 2; rrp47Δ (YF1465), lane 3; WT (YF1444), lane 4; rrp6Δ (YSB2244), lanes 5 and 6; WT (YF1977), lane 7; dis3 (D171A) (YF1978), lane 8 and dis3 (D551N) (YF1979), lane 9. RNA was resolved on a 1.2% agarose gel and probed for TLC1 RNA. 25 and 18S rRNAs are shown below. Numbers below indicate fold increase in poly(A)+, poly(A) − TLC1 RNA levels relative to WT and poly(A)+/poly(A) − ratio. D) Accumulation of poly(A)+ TER1 RNA in S. pombe exosome mutant strains. Northern blot analysis of total RNA, lanes 1, 3 and 5, or oligo-dT purified RNA, lanes 2, 4 and 6 from WT (YP34), lanes 1 and 2; rrp6Δ (YP35), lanes 3 and 4; dis3-54 (YP50), lanes 5 and 6. RNA was resolved on a 5% PAGE and TER1 RNA was visualized with the probe depicted in (A). Positions of poly(A)+ and poly(A) − TER1 RNAs are indicated with grey boxes. 18S rRNA is shown below. Numbers below indicate fold increase in RNA levels relative to WT.

Mentions: Mature functional sn/snoRNAs are believed to be produced from polyadenylated RNA precursors by a process that involves trimming by the exosome complex in the nucleus. Accumulation of the 3′-extended RNA species is typically observed in exosome mutants, however this does not always correlate with a decrease in the mature RNA levels, perhaps due to the redundancy between different exosome subunits or the presence of other processing pathway(s) that function in addition to the exosome complex [6], [29], [30], [31]. Moreover, for some RNAs (such as U4 and U5) increased levels of the mature RNA are observed in exosome mutants suggesting that the exosome complex contributes to the turn-over of the mature species of these RNAs [29]. Similarity in the organization of telomerase RNA and snRNAs suggests that telomerase RNA might be processed by a similar mechanism. To test this hypothesis we compared the levels of TLC1 in wild type (WT) cells and exosome mutants by northern blotting. In agreement with published data [12], two forms of telomerase RNA were observed: the predominant shorter ‘mature’ form and a longer minor form (Figure 1B, lanes 1 and 3). The longer form represents ∼10–15% of TLC1 RNA in WT cells but is enriched upon oligo-dT purification, consistent with previous report that it is polyadenylated [12] (Figure 1C, lane 2). Interestingly, in exosome mutants lacking exonucleolytic activity (rrp6Δ and dis3 exo- (catalytic site inactivating D551N mutation)) an accumulation of the poly(A)+ form of TLC1 is observed (Figure 1B lane 4; C lanes 5,6 and 9) together with a slight increase in levels of the mature RNA. In contrast, a mutation abolishing the endonucleolytic activity of Dis3 (D171A) shows no effect on TLC1 RNA levels (Figure 1C, lane 8). Consistent with its proposed role in assisting Rrp6 with the processing of structured RNAs, the Rrp47 mutant displays a substantial increase in poly(A)+ TLC1 RNA (Figure 1B, lane 2 and C, lane 3). Surprisingly, however, the ratio between longer and shorter forms of TLC1 is more dramatically affected in rrp47Δ and dis3 exo- mutants than in the rrp6Δ strain (Figure 1B, compare lanes 2 and 4; C compare lanes 3, 5 and 9). This suggests that in addition to its known function as a cofactor for Rrp6, Rrp47 may serve as a cofactor for the exosome core complex. We next investigated the effect of metabolic depletion of Nrd1, a protein involved in transcription termination and in exosome recruitment to the 3′ termini of pol II transcribed ncRNAs [17]. For this experiment Nrd1 was expressed from a galactose inducible promoter such that expression could be turned off by growing cells on glucose. As expected, upon Nrd1 depletion TLC1 levels are increased (Figure S1, compare lanes 1 and 3), consistent with a role for the exosome in affecting levels of TLC1 RNA. Furthermore, an additional increase in TLC1 levels is observed when Nrd1 depletion is combined with a deletion of Rrp47 (Figure S1, lane 7). It was recently reported that NrdΔ1 is involved in transcription termination of this non-coding transcript and therefore presence of poly(A)+ is partially dependent on the functional Nrd1 termination pathway [18]. This could possibly explain why we do not observe accumulation of poly(A)+ form in the absence of Nrd1.


The Sm complex is required for the processing of non-coding RNAs by the exosome.

Coy S, Volanakis A, Shah S, Vasiljeva L - PLoS ONE (2013)

Exosome mutants accumulate poly(A)+ telomerase RNA.A) Schematic diagram describing the positions of TLC1 (S. cerevisiae) and TER1 (S. pombe) probes for telomerase RNA. Probes are depicted as black bars. B) Accumulation of poly(A)+ TLC1 RNA in S. cerevisiae exosome mutant strains. Northern blot analysis of total RNA from WT (YF336), lane 1; rrp47Δ (YF1465), lane 2; WT (YF1444), lane 3 and rrp6Δ (YSB2244), lane 4. RNA was resolved on a 5% PAGE and TLC1 RNA was visualized with the probe depicted in (A). Positions of poly(A)+ and poly(A)− TLC1 RNAs are indicated with grey boxes. Methylene-Blue stained 18 and 25S rRNAs are shown below and serve as a loading control. C) Exonucleolytic activity of the exosome is required for the processing of poly(A)+ TLC1 RNA. Northern blot analysis of total RNA, lanes 1, 3–5 and 7–9 or oligo-dT purified RNA, lanes 2 and 6. RNA was isolated from WT (YF336), lanes 1 and 2; rrp47Δ (YF1465), lane 3; WT (YF1444), lane 4; rrp6Δ (YSB2244), lanes 5 and 6; WT (YF1977), lane 7; dis3 (D171A) (YF1978), lane 8 and dis3 (D551N) (YF1979), lane 9. RNA was resolved on a 1.2% agarose gel and probed for TLC1 RNA. 25 and 18S rRNAs are shown below. Numbers below indicate fold increase in poly(A)+, poly(A) − TLC1 RNA levels relative to WT and poly(A)+/poly(A) − ratio. D) Accumulation of poly(A)+ TER1 RNA in S. pombe exosome mutant strains. Northern blot analysis of total RNA, lanes 1, 3 and 5, or oligo-dT purified RNA, lanes 2, 4 and 6 from WT (YP34), lanes 1 and 2; rrp6Δ (YP35), lanes 3 and 4; dis3-54 (YP50), lanes 5 and 6. RNA was resolved on a 5% PAGE and TER1 RNA was visualized with the probe depicted in (A). Positions of poly(A)+ and poly(A) − TER1 RNAs are indicated with grey boxes. 18S rRNA is shown below. Numbers below indicate fold increase in RNA levels relative to WT.
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Related In: Results  -  Collection

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pone-0065606-g001: Exosome mutants accumulate poly(A)+ telomerase RNA.A) Schematic diagram describing the positions of TLC1 (S. cerevisiae) and TER1 (S. pombe) probes for telomerase RNA. Probes are depicted as black bars. B) Accumulation of poly(A)+ TLC1 RNA in S. cerevisiae exosome mutant strains. Northern blot analysis of total RNA from WT (YF336), lane 1; rrp47Δ (YF1465), lane 2; WT (YF1444), lane 3 and rrp6Δ (YSB2244), lane 4. RNA was resolved on a 5% PAGE and TLC1 RNA was visualized with the probe depicted in (A). Positions of poly(A)+ and poly(A)− TLC1 RNAs are indicated with grey boxes. Methylene-Blue stained 18 and 25S rRNAs are shown below and serve as a loading control. C) Exonucleolytic activity of the exosome is required for the processing of poly(A)+ TLC1 RNA. Northern blot analysis of total RNA, lanes 1, 3–5 and 7–9 or oligo-dT purified RNA, lanes 2 and 6. RNA was isolated from WT (YF336), lanes 1 and 2; rrp47Δ (YF1465), lane 3; WT (YF1444), lane 4; rrp6Δ (YSB2244), lanes 5 and 6; WT (YF1977), lane 7; dis3 (D171A) (YF1978), lane 8 and dis3 (D551N) (YF1979), lane 9. RNA was resolved on a 1.2% agarose gel and probed for TLC1 RNA. 25 and 18S rRNAs are shown below. Numbers below indicate fold increase in poly(A)+, poly(A) − TLC1 RNA levels relative to WT and poly(A)+/poly(A) − ratio. D) Accumulation of poly(A)+ TER1 RNA in S. pombe exosome mutant strains. Northern blot analysis of total RNA, lanes 1, 3 and 5, or oligo-dT purified RNA, lanes 2, 4 and 6 from WT (YP34), lanes 1 and 2; rrp6Δ (YP35), lanes 3 and 4; dis3-54 (YP50), lanes 5 and 6. RNA was resolved on a 5% PAGE and TER1 RNA was visualized with the probe depicted in (A). Positions of poly(A)+ and poly(A) − TER1 RNAs are indicated with grey boxes. 18S rRNA is shown below. Numbers below indicate fold increase in RNA levels relative to WT.
Mentions: Mature functional sn/snoRNAs are believed to be produced from polyadenylated RNA precursors by a process that involves trimming by the exosome complex in the nucleus. Accumulation of the 3′-extended RNA species is typically observed in exosome mutants, however this does not always correlate with a decrease in the mature RNA levels, perhaps due to the redundancy between different exosome subunits or the presence of other processing pathway(s) that function in addition to the exosome complex [6], [29], [30], [31]. Moreover, for some RNAs (such as U4 and U5) increased levels of the mature RNA are observed in exosome mutants suggesting that the exosome complex contributes to the turn-over of the mature species of these RNAs [29]. Similarity in the organization of telomerase RNA and snRNAs suggests that telomerase RNA might be processed by a similar mechanism. To test this hypothesis we compared the levels of TLC1 in wild type (WT) cells and exosome mutants by northern blotting. In agreement with published data [12], two forms of telomerase RNA were observed: the predominant shorter ‘mature’ form and a longer minor form (Figure 1B, lanes 1 and 3). The longer form represents ∼10–15% of TLC1 RNA in WT cells but is enriched upon oligo-dT purification, consistent with previous report that it is polyadenylated [12] (Figure 1C, lane 2). Interestingly, in exosome mutants lacking exonucleolytic activity (rrp6Δ and dis3 exo- (catalytic site inactivating D551N mutation)) an accumulation of the poly(A)+ form of TLC1 is observed (Figure 1B lane 4; C lanes 5,6 and 9) together with a slight increase in levels of the mature RNA. In contrast, a mutation abolishing the endonucleolytic activity of Dis3 (D171A) shows no effect on TLC1 RNA levels (Figure 1C, lane 8). Consistent with its proposed role in assisting Rrp6 with the processing of structured RNAs, the Rrp47 mutant displays a substantial increase in poly(A)+ TLC1 RNA (Figure 1B, lane 2 and C, lane 3). Surprisingly, however, the ratio between longer and shorter forms of TLC1 is more dramatically affected in rrp47Δ and dis3 exo- mutants than in the rrp6Δ strain (Figure 1B, compare lanes 2 and 4; C compare lanes 3, 5 and 9). This suggests that in addition to its known function as a cofactor for Rrp6, Rrp47 may serve as a cofactor for the exosome core complex. We next investigated the effect of metabolic depletion of Nrd1, a protein involved in transcription termination and in exosome recruitment to the 3′ termini of pol II transcribed ncRNAs [17]. For this experiment Nrd1 was expressed from a galactose inducible promoter such that expression could be turned off by growing cells on glucose. As expected, upon Nrd1 depletion TLC1 levels are increased (Figure S1, compare lanes 1 and 3), consistent with a role for the exosome in affecting levels of TLC1 RNA. Furthermore, an additional increase in TLC1 levels is observed when Nrd1 depletion is combined with a deletion of Rrp47 (Figure S1, lane 7). It was recently reported that NrdΔ1 is involved in transcription termination of this non-coding transcript and therefore presence of poly(A)+ is partially dependent on the functional Nrd1 termination pathway [18]. This could possibly explain why we do not observe accumulation of poly(A)+ form in the absence of Nrd1.

Bottom Line: Additionally, we demonstrate that the same pathway is involved in the maturation of snRNAs.We also show that telomerase RNA accumulates in Schizosaccharomyces pombe exosome mutants, suggesting a conserved role for the exosome in processing and degradation of telomerase RNA.In summary, our data provide important mechanistic insight into the regulation of exosome dependent RNA processing as well as telomerase RNA biogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Oxford, Oxford, United Kingdom.

ABSTRACT
A key question in the field of RNA regulation is how some exosome substrates, such as spliceosomal snRNAs and telomerase RNA, evade degradation and are processed into stable, functional RNA molecules. Typical feature of these non-coding RNAs is presence of the Sm complex at the 3'end of the mature RNA molecule. Here, we report that in Saccharomyces cerevisiae presence of intact Sm binding site is required for the exosome-mediated processing of telomerase RNA from a polyadenylated precursor into its mature form and is essential for its function in elongating telomeres. Additionally, we demonstrate that the same pathway is involved in the maturation of snRNAs. Furthermore, the insertion of an Sm binding site into an unstable RNA that is normally completely destroyed by the exosome, leads to its partial stabilization. We also show that telomerase RNA accumulates in Schizosaccharomyces pombe exosome mutants, suggesting a conserved role for the exosome in processing and degradation of telomerase RNA. In summary, our data provide important mechanistic insight into the regulation of exosome dependent RNA processing as well as telomerase RNA biogenesis.

Show MeSH
Related in: MedlinePlus