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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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Telomerase RNA can be trimmed by the exosome complex in vitro.A) Schematic diagram explaining the experimental set up of the in vitro telomerase RNA processing reaction. Exosome complex purified as described in (A) was incubated with the Est2 associated telomerase RNA isolated on IgG agarose from cells expressing ProteinA-Est2 (YLV124, 125, and 126 strains). B) The exosome processes but does not degrade the longer form of telomerase RNA in the presence of a functional Sm site in vitro. Telomerase RNA purified from rrp47Δ (YLV124), tlc1sm4C5C, rrp47Δ (YLV125) and TLC1 (YLV126) was incubated with the exosome complex for 90 min and reaction products were analysed by RT-PCR using oligos F (#2492) and R1 (#2489) (lanes 2–7), F and R2 (#2493) (lanes 8–14). PCR conditions were optimized and reactions were performed for 25 cycles, PCR products were resolved on a 2% agarose gel. C, D) Relative RNA levels were measured by quantification of PCR band intensities from lanes 2–7 and 8–13 in (B) using ImageJ v1.32 software. Error bars are from three independent repetitions.
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pone-0065606-g004: Telomerase RNA can be trimmed by the exosome complex in vitro.A) Schematic diagram explaining the experimental set up of the in vitro telomerase RNA processing reaction. Exosome complex purified as described in (A) was incubated with the Est2 associated telomerase RNA isolated on IgG agarose from cells expressing ProteinA-Est2 (YLV124, 125, and 126 strains). B) The exosome processes but does not degrade the longer form of telomerase RNA in the presence of a functional Sm site in vitro. Telomerase RNA purified from rrp47Δ (YLV124), tlc1sm4C5C, rrp47Δ (YLV125) and TLC1 (YLV126) was incubated with the exosome complex for 90 min and reaction products were analysed by RT-PCR using oligos F (#2492) and R1 (#2489) (lanes 2–7), F and R2 (#2493) (lanes 8–14). PCR conditions were optimized and reactions were performed for 25 cycles, PCR products were resolved on a 2% agarose gel. C, D) Relative RNA levels were measured by quantification of PCR band intensities from lanes 2–7 and 8–13 in (B) using ImageJ v1.32 software. Error bars are from three independent repetitions.

Mentions: To assay the ability of the exosome core to either degrade or process the different forms of telomerase RNA, Est2-bound TLC1 RNA was purified from rrp47Δ, tlc1-sm4C5C rrp47Δ and WT strains and incubated with the purified exosome (Figure 4A). Levels of TLC1 RNA before and after incubation of the native telomerase complex with the exosome were analyzed by RT-PCR (Figure 4B and C). The primer pairs used for RT-PCR are designed to either amplify specifically the longer form (F and R1) or a region of TLC1 upstream of the Sm site (F and R2). Thus primer pair F/R2 amplifies both forms and can be used to measure the total levels of TLC1 RNA. While total levels of TLC1 associated with Est2 are the same for all three purifications (Figure 4B, lanes 9, 11 and 13; and Figure 4C), levels of the longer TLC1 form significantly vary depending on which strain the telomerase complex was purified from (Figure 4B, lanes 3, 5 and 7; and Figure 4C). In WT cells, very low levels of the longer TLC1 form associated with Est2 reflecting the low abundance of this form of RNA in the cell (Figure 4B, lane 7). Upon mutation of the exosome (rrp47Δ), much higher levels of the long form are detected to be a part of the telomerase complex (Figure 4B, lanes 3 and 5), correlating with the observed increase in abundance of this form observed by northern blotting (Figure 2B). As expected, levels of the longer TLC1 form are decreased upon incubation of the Est2 bound RNA with the exosome complex (Figure 4B, compare lanes 2, 4 and 6 to lanes 3, 5 and 7; and Figure 4C). Importantly, for TLC1 carrying a mutated Sm site, this correlates with a decrease in the overall TLC1 level indicating that the entire RNA is degraded by the exosome when the Sm comlex is not bound (Figure 4B, compare lanes 10 and 11; and Figure 4C). In contrast, the overall levels of TLC1 with a functional Sm site remain the same upon incubation with the exosome (Figure 4B, compare lanes 8 and 9; and Figure 4D). These results demonstrate that telomerase RNA upstream of the Sm site is more resistant to exosome mediated degradation compared to the part of the RNA down-stream of the Sm site. This data are in support of a model where the exosome complex trims the telomerase poly(A)+ RNA. However, we cannot rule out a possibility that a fraction of poly(A)+ TLC1 RNA can also be turned over by the exosome complex in vivo.


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)

Telomerase RNA can be trimmed by the exosome complex in vitro.A) Schematic diagram explaining the experimental set up of the in vitro telomerase RNA processing reaction. Exosome complex purified as described in (A) was incubated with the Est2 associated telomerase RNA isolated on IgG agarose from cells expressing ProteinA-Est2 (YLV124, 125, and 126 strains). B) The exosome processes but does not degrade the longer form of telomerase RNA in the presence of a functional Sm site in vitro. Telomerase RNA purified from rrp47Δ (YLV124), tlc1sm4C5C, rrp47Δ (YLV125) and TLC1 (YLV126) was incubated with the exosome complex for 90 min and reaction products were analysed by RT-PCR using oligos F (#2492) and R1 (#2489) (lanes 2–7), F and R2 (#2493) (lanes 8–14). PCR conditions were optimized and reactions were performed for 25 cycles, PCR products were resolved on a 2% agarose gel. C, D) Relative RNA levels were measured by quantification of PCR band intensities from lanes 2–7 and 8–13 in (B) using ImageJ v1.32 software. Error bars are from three independent repetitions.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3675052&req=5

pone-0065606-g004: Telomerase RNA can be trimmed by the exosome complex in vitro.A) Schematic diagram explaining the experimental set up of the in vitro telomerase RNA processing reaction. Exosome complex purified as described in (A) was incubated with the Est2 associated telomerase RNA isolated on IgG agarose from cells expressing ProteinA-Est2 (YLV124, 125, and 126 strains). B) The exosome processes but does not degrade the longer form of telomerase RNA in the presence of a functional Sm site in vitro. Telomerase RNA purified from rrp47Δ (YLV124), tlc1sm4C5C, rrp47Δ (YLV125) and TLC1 (YLV126) was incubated with the exosome complex for 90 min and reaction products were analysed by RT-PCR using oligos F (#2492) and R1 (#2489) (lanes 2–7), F and R2 (#2493) (lanes 8–14). PCR conditions were optimized and reactions were performed for 25 cycles, PCR products were resolved on a 2% agarose gel. C, D) Relative RNA levels were measured by quantification of PCR band intensities from lanes 2–7 and 8–13 in (B) using ImageJ v1.32 software. Error bars are from three independent repetitions.
Mentions: To assay the ability of the exosome core to either degrade or process the different forms of telomerase RNA, Est2-bound TLC1 RNA was purified from rrp47Δ, tlc1-sm4C5C rrp47Δ and WT strains and incubated with the purified exosome (Figure 4A). Levels of TLC1 RNA before and after incubation of the native telomerase complex with the exosome were analyzed by RT-PCR (Figure 4B and C). The primer pairs used for RT-PCR are designed to either amplify specifically the longer form (F and R1) or a region of TLC1 upstream of the Sm site (F and R2). Thus primer pair F/R2 amplifies both forms and can be used to measure the total levels of TLC1 RNA. While total levels of TLC1 associated with Est2 are the same for all three purifications (Figure 4B, lanes 9, 11 and 13; and Figure 4C), levels of the longer TLC1 form significantly vary depending on which strain the telomerase complex was purified from (Figure 4B, lanes 3, 5 and 7; and Figure 4C). In WT cells, very low levels of the longer TLC1 form associated with Est2 reflecting the low abundance of this form of RNA in the cell (Figure 4B, lane 7). Upon mutation of the exosome (rrp47Δ), much higher levels of the long form are detected to be a part of the telomerase complex (Figure 4B, lanes 3 and 5), correlating with the observed increase in abundance of this form observed by northern blotting (Figure 2B). As expected, levels of the longer TLC1 form are decreased upon incubation of the Est2 bound RNA with the exosome complex (Figure 4B, compare lanes 2, 4 and 6 to lanes 3, 5 and 7; and Figure 4C). Importantly, for TLC1 carrying a mutated Sm site, this correlates with a decrease in the overall TLC1 level indicating that the entire RNA is degraded by the exosome when the Sm comlex is not bound (Figure 4B, compare lanes 10 and 11; and Figure 4C). In contrast, the overall levels of TLC1 with a functional Sm site remain the same upon incubation with the exosome (Figure 4B, compare lanes 8 and 9; and Figure 4D). These results demonstrate that telomerase RNA upstream of the Sm site is more resistant to exosome mediated degradation compared to the part of the RNA down-stream of the Sm site. This data are in support of a model where the exosome complex trims the telomerase poly(A)+ RNA. However, we cannot rule out a possibility that a fraction of poly(A)+ TLC1 RNA can also be turned over by the exosome complex in vivo.

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