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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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An Sm site is required for the processing of the U1 RNA precursor by the exosome complex.A) Schematic diagram describing the organization of plasmid-borne SNR19 and SNR19Δ192–507 genes, which drive the expression of U1 and U1Δ192–507 RNA respectively. Endogenous SNR19 was deleted and replaced by plasmids encoding SNR19 and either SNR19Δ192–507 or SNR19Δ192–507sm, that lacks an Sm site. The plasmid-borne copy of full length SNR19 is under the control of a galactose inducible promoter (GAL10p), such that expression of the WT gene can be shut down by culturing cells in glucose media, while SNR19Δ192–507 and SNR19Δ192–507sm are expressed constitutively from the endogenous SNR19 promoter (SNR19p). B) Processing of U1 RNA upon disruption of the exosome/Sm-mediated processing pathway. Northern blot analysis of U1 processing in U1(Δ192–507)sm, GAL::U1 (YF2081); U1(Δ192–507)sm, GAL::U1, rrp47Δ (YLV48); U1(Δ192–507) (YF2088) and U1(Δ192–507), rrp47Δ (YLV68) (lanes 1–4 respectively). Positions of U1(Δ192–507) precursor and U1(Δ192–507) mature RNAs are indicated. Bands corresponding to the precursor RNA are marked with asterisks. 18S RNA is also shown. C) U1 precursor RNA does not compensate for the function of mature U1 RNA in pre-mRNA splicing. Splicing of RP51A RNA was analyzed by northern blot of total RNA from U1(Δ192–507)sm, GAL::U1 (YF2081); or U1(Δ192–507)sm, GAL::U1, rrp47Δ (YLV48). Cells were pre-grown on galactose media to OD = 0.5 to allow for expression of full length U1 from the GAL10 promoter (shown in (A)). Logarithmic cultures were maintained for a further 10 hours in media containing galactose (lanes 1 and 3) or glucose (lanes 2 and 4). Positions of spliced RP51A and its un-spliced precursor are indicated. Both forms of RP51A RNA (spliced and un-spliced) appear as double bands as previously reported. To control for loading, RNA was also probed for ADH1. D) Quantification of splicing efficiency of Sm mutated U1 RNA relative to WT. The splicing efficiency observed in the experiments described in Figure 5C was calculated as a ratio of spliced/un-spliced RP51A RNA precursor and normalized for the WT value.
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pone-0065606-g005: An Sm site is required for the processing of the U1 RNA precursor by the exosome complex.A) Schematic diagram describing the organization of plasmid-borne SNR19 and SNR19Δ192–507 genes, which drive the expression of U1 and U1Δ192–507 RNA respectively. Endogenous SNR19 was deleted and replaced by plasmids encoding SNR19 and either SNR19Δ192–507 or SNR19Δ192–507sm, that lacks an Sm site. The plasmid-borne copy of full length SNR19 is under the control of a galactose inducible promoter (GAL10p), such that expression of the WT gene can be shut down by culturing cells in glucose media, while SNR19Δ192–507 and SNR19Δ192–507sm are expressed constitutively from the endogenous SNR19 promoter (SNR19p). B) Processing of U1 RNA upon disruption of the exosome/Sm-mediated processing pathway. Northern blot analysis of U1 processing in U1(Δ192–507)sm, GAL::U1 (YF2081); U1(Δ192–507)sm, GAL::U1, rrp47Δ (YLV48); U1(Δ192–507) (YF2088) and U1(Δ192–507), rrp47Δ (YLV68) (lanes 1–4 respectively). Positions of U1(Δ192–507) precursor and U1(Δ192–507) mature RNAs are indicated. Bands corresponding to the precursor RNA are marked with asterisks. 18S RNA is also shown. C) U1 precursor RNA does not compensate for the function of mature U1 RNA in pre-mRNA splicing. Splicing of RP51A RNA was analyzed by northern blot of total RNA from U1(Δ192–507)sm, GAL::U1 (YF2081); or U1(Δ192–507)sm, GAL::U1, rrp47Δ (YLV48). Cells were pre-grown on galactose media to OD = 0.5 to allow for expression of full length U1 from the GAL10 promoter (shown in (A)). Logarithmic cultures were maintained for a further 10 hours in media containing galactose (lanes 1 and 3) or glucose (lanes 2 and 4). Positions of spliced RP51A and its un-spliced precursor are indicated. Both forms of RP51A RNA (spliced and un-spliced) appear as double bands as previously reported. To control for loading, RNA was also probed for ADH1. D) Quantification of splicing efficiency of Sm mutated U1 RNA relative to WT. The splicing efficiency observed in the experiments described in Figure 5C was calculated as a ratio of spliced/un-spliced RP51A RNA precursor and normalized for the WT value.

Mentions: U1 RNA is representative of snRNAs, containing a 9 nucleotide Sm site upstream of its mature 3′ end (position 553–559) (Figure S4). Since U1 is known to be trimmed by the exosome complex following RNAse III (Rnt1) cleavage [20], [29] we next investigated the influence of the Sm site on this process. Replacement of the natural Sm site (AAUUUUUGA) with a non-binding sequence (AAUUCACAC) was previously reported to result in the disappearance of mature U1 RNA, leading to defective mRNA splicing and lethality [20]. To support cell viability, we used a previously described system [20], whereby plasmid encoded WT GAL10-driven U1 RNA was expressed in a U1 deletion strain. The Sm site replacement sequence was introduced into a constitutively expressed plasmid-encoded U1 derivative (U1Δ192–507) allowing for distinction from full size WT U1 RNA (Figure 5A) [20]. In these strains, when cells are grown on galactose, GAL10-driven U1 RNA is functional and processed properly [46], [47]. Processing of U1Δ192–507 and U1Δ192–507sm was studied with and without functional Rrp47 (Figure 5B). In agreement with the previous report, in the presence of functional exosome U1Δ192–507 was fully processed into a shorter mature RNA (Figure 5B, lane 3). As expected, some accumulation of the unprocessed precursor was observed in the rrp47Δ mutant (Figure 5B, lane 4). Consistent with our data on TLC1 processing, upon mutation of the Sm site the mature form of U1Δ192–507 was no longer detected, instead a slight accumulation of the precursor RNA was observed (Figure 5B, compare lanes 1 and 3). Deletion of RRP47 from the Sm mutant strain resulted in an increase in the abundance of U1 precursor RNA, whilst still no mature form was observed (Figure 5B, lane 2). The additional increase in abundance of the U1 precursor in the double mutant (U1Δ192-507sm/rrp47Δ) compared to rrp47Δ alone (Figure 5B, compare lanes 2 and 4) probably reflects some functional redundancy between Rrp6 and the core of the exosome. The accumulation of the 3′-extended U1 precursor upon mutating either the Sm site or the exosome suggests that as is the case for TLC1, both components are required for the processing of U1 precursor RNA.


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)

An Sm site is required for the processing of the U1 RNA precursor by the exosome complex.A) Schematic diagram describing the organization of plasmid-borne SNR19 and SNR19Δ192–507 genes, which drive the expression of U1 and U1Δ192–507 RNA respectively. Endogenous SNR19 was deleted and replaced by plasmids encoding SNR19 and either SNR19Δ192–507 or SNR19Δ192–507sm, that lacks an Sm site. The plasmid-borne copy of full length SNR19 is under the control of a galactose inducible promoter (GAL10p), such that expression of the WT gene can be shut down by culturing cells in glucose media, while SNR19Δ192–507 and SNR19Δ192–507sm are expressed constitutively from the endogenous SNR19 promoter (SNR19p). B) Processing of U1 RNA upon disruption of the exosome/Sm-mediated processing pathway. Northern blot analysis of U1 processing in U1(Δ192–507)sm, GAL::U1 (YF2081); U1(Δ192–507)sm, GAL::U1, rrp47Δ (YLV48); U1(Δ192–507) (YF2088) and U1(Δ192–507), rrp47Δ (YLV68) (lanes 1–4 respectively). Positions of U1(Δ192–507) precursor and U1(Δ192–507) mature RNAs are indicated. Bands corresponding to the precursor RNA are marked with asterisks. 18S RNA is also shown. C) U1 precursor RNA does not compensate for the function of mature U1 RNA in pre-mRNA splicing. Splicing of RP51A RNA was analyzed by northern blot of total RNA from U1(Δ192–507)sm, GAL::U1 (YF2081); or U1(Δ192–507)sm, GAL::U1, rrp47Δ (YLV48). Cells were pre-grown on galactose media to OD = 0.5 to allow for expression of full length U1 from the GAL10 promoter (shown in (A)). Logarithmic cultures were maintained for a further 10 hours in media containing galactose (lanes 1 and 3) or glucose (lanes 2 and 4). Positions of spliced RP51A and its un-spliced precursor are indicated. Both forms of RP51A RNA (spliced and un-spliced) appear as double bands as previously reported. To control for loading, RNA was also probed for ADH1. D) Quantification of splicing efficiency of Sm mutated U1 RNA relative to WT. The splicing efficiency observed in the experiments described in Figure 5C was calculated as a ratio of spliced/un-spliced RP51A RNA precursor and normalized for the WT value.
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Related In: Results  -  Collection

Show All Figures
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pone-0065606-g005: An Sm site is required for the processing of the U1 RNA precursor by the exosome complex.A) Schematic diagram describing the organization of plasmid-borne SNR19 and SNR19Δ192–507 genes, which drive the expression of U1 and U1Δ192–507 RNA respectively. Endogenous SNR19 was deleted and replaced by plasmids encoding SNR19 and either SNR19Δ192–507 or SNR19Δ192–507sm, that lacks an Sm site. The plasmid-borne copy of full length SNR19 is under the control of a galactose inducible promoter (GAL10p), such that expression of the WT gene can be shut down by culturing cells in glucose media, while SNR19Δ192–507 and SNR19Δ192–507sm are expressed constitutively from the endogenous SNR19 promoter (SNR19p). B) Processing of U1 RNA upon disruption of the exosome/Sm-mediated processing pathway. Northern blot analysis of U1 processing in U1(Δ192–507)sm, GAL::U1 (YF2081); U1(Δ192–507)sm, GAL::U1, rrp47Δ (YLV48); U1(Δ192–507) (YF2088) and U1(Δ192–507), rrp47Δ (YLV68) (lanes 1–4 respectively). Positions of U1(Δ192–507) precursor and U1(Δ192–507) mature RNAs are indicated. Bands corresponding to the precursor RNA are marked with asterisks. 18S RNA is also shown. C) U1 precursor RNA does not compensate for the function of mature U1 RNA in pre-mRNA splicing. Splicing of RP51A RNA was analyzed by northern blot of total RNA from U1(Δ192–507)sm, GAL::U1 (YF2081); or U1(Δ192–507)sm, GAL::U1, rrp47Δ (YLV48). Cells were pre-grown on galactose media to OD = 0.5 to allow for expression of full length U1 from the GAL10 promoter (shown in (A)). Logarithmic cultures were maintained for a further 10 hours in media containing galactose (lanes 1 and 3) or glucose (lanes 2 and 4). Positions of spliced RP51A and its un-spliced precursor are indicated. Both forms of RP51A RNA (spliced and un-spliced) appear as double bands as previously reported. To control for loading, RNA was also probed for ADH1. D) Quantification of splicing efficiency of Sm mutated U1 RNA relative to WT. The splicing efficiency observed in the experiments described in Figure 5C was calculated as a ratio of spliced/un-spliced RP51A RNA precursor and normalized for the WT value.
Mentions: U1 RNA is representative of snRNAs, containing a 9 nucleotide Sm site upstream of its mature 3′ end (position 553–559) (Figure S4). Since U1 is known to be trimmed by the exosome complex following RNAse III (Rnt1) cleavage [20], [29] we next investigated the influence of the Sm site on this process. Replacement of the natural Sm site (AAUUUUUGA) with a non-binding sequence (AAUUCACAC) was previously reported to result in the disappearance of mature U1 RNA, leading to defective mRNA splicing and lethality [20]. To support cell viability, we used a previously described system [20], whereby plasmid encoded WT GAL10-driven U1 RNA was expressed in a U1 deletion strain. The Sm site replacement sequence was introduced into a constitutively expressed plasmid-encoded U1 derivative (U1Δ192–507) allowing for distinction from full size WT U1 RNA (Figure 5A) [20]. In these strains, when cells are grown on galactose, GAL10-driven U1 RNA is functional and processed properly [46], [47]. Processing of U1Δ192–507 and U1Δ192–507sm was studied with and without functional Rrp47 (Figure 5B). In agreement with the previous report, in the presence of functional exosome U1Δ192–507 was fully processed into a shorter mature RNA (Figure 5B, lane 3). As expected, some accumulation of the unprocessed precursor was observed in the rrp47Δ mutant (Figure 5B, lane 4). Consistent with our data on TLC1 processing, upon mutation of the Sm site the mature form of U1Δ192–507 was no longer detected, instead a slight accumulation of the precursor RNA was observed (Figure 5B, compare lanes 1 and 3). Deletion of RRP47 from the Sm mutant strain resulted in an increase in the abundance of U1 precursor RNA, whilst still no mature form was observed (Figure 5B, lane 2). The additional increase in abundance of the U1 precursor in the double mutant (U1Δ192-507sm/rrp47Δ) compared to rrp47Δ alone (Figure 5B, compare lanes 2 and 4) probably reflects some functional redundancy between Rrp6 and the core of the exosome. The accumulation of the 3′-extended U1 precursor upon mutating either the Sm site or the exosome suggests that as is the case for TLC1, both components are required for the processing of U1 precursor RNA.

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