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Depletion of key protein components of the RISC pathway impairs pre-ribosomal RNA processing.

Liang XH, Crooke ST - Nucleic Acids Res. (2011)

Bottom Line: Here, we show that depletion of key proteins of the RISC pathway by antisense oligonucleotides significantly impairs pre-rRNA processing in human cells.Both Dicer and Ago2 were detected in the nuclear fraction, and reduction of Dicer altered the structure of the nucleolus, where pre-rRNA processing occurs.Together, these results suggest that Drosha and Dicer are implicated in rRNA biogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Core Antisense Research, ISIS Pharmaceuticals, Inc., 1896 Rutherford Rd, Carlsbad, CA 92008, USA. lliang@isisph.com

ABSTRACT
Little is known about whether components of the RNA-induced silencing complex (RISC) mediate the biogenesis of RNAs other than miRNA. Here, we show that depletion of key proteins of the RISC pathway by antisense oligonucleotides significantly impairs pre-rRNA processing in human cells. In cells depleted of Drosha or Dicer, different precursors to 5.8S rRNA strongly accumulated, without affecting normal endonucleolytic cleavages. Moderate yet distinct processing defects were also observed in Ago2-depleted cells. Physical links between pre-rRNA and these proteins were identified by co-immunoprecipitation analyses. Interestingly, simultaneous depletion of Dicer and Drosha led to a different processing defect, causing slower production of 28S rRNA and its precursor. Both Dicer and Ago2 were detected in the nuclear fraction, and reduction of Dicer altered the structure of the nucleolus, where pre-rRNA processing occurs. Together, these results suggest that Drosha and Dicer are implicated in rRNA biogenesis.

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Pre-rRNA accumulation in cells depleted of RISC pathway proteins. (A) Pre-rRNA processing pathway in mammals. ETS and ITS are external and internal transcribed spacers, respectively. The position of the hybridization probe used in (D and E) is shown as a solid bar above 47S pre-rRNA. (B) mRNA levels were dramatically reduced 48 h after treatment with 50 nM ASOs targeting Drosha (ISIS25690), Ago2 (ISIS136764) or Dicer (ISIS138648), as determined by qRT–PCR. The error bars indicate standard deviation from two independent experiments with three replicates. UTC, untreated cells; +ASO, cells treated with ASOs. (C) The levels of targeted proteins were significantly reduced by ASO treatment, as determined by western analysis. Alpha-tubulin was used as a control for loading. (D) Northern hybridization for pre-rRNA species using a probe specific to the boundary of 5.8S rRNA/ITS2. Total RNA prepared from test cells 48 h after ASO treatment was separated on a 1.2% agarose gel, and the blot was subjected to hybridization. The arrows indicate precursors to 5.8S rRNA. The positions of mature rRNAs are indicated. Lower panel shows ethidium bromide staining of rRNAs in the same gel. (E) Pre-5.8S rRNA accumulated in cells depleted of Drosha, Ago2 or Dicer. Total RNA as used in (C) was separated in an 8% polyacrylamide, 7M urea gel and the blot was hybridized using the same probe as in (D). The arrows indicate different pre-5.8S rRNA species (marked as A, B and C). U3 snoRNA was probed to serve as a loading control. Lower panel shows ethidium bromide staining of rRNAs in the same gel.
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Figure 1: Pre-rRNA accumulation in cells depleted of RISC pathway proteins. (A) Pre-rRNA processing pathway in mammals. ETS and ITS are external and internal transcribed spacers, respectively. The position of the hybridization probe used in (D and E) is shown as a solid bar above 47S pre-rRNA. (B) mRNA levels were dramatically reduced 48 h after treatment with 50 nM ASOs targeting Drosha (ISIS25690), Ago2 (ISIS136764) or Dicer (ISIS138648), as determined by qRT–PCR. The error bars indicate standard deviation from two independent experiments with three replicates. UTC, untreated cells; +ASO, cells treated with ASOs. (C) The levels of targeted proteins were significantly reduced by ASO treatment, as determined by western analysis. Alpha-tubulin was used as a control for loading. (D) Northern hybridization for pre-rRNA species using a probe specific to the boundary of 5.8S rRNA/ITS2. Total RNA prepared from test cells 48 h after ASO treatment was separated on a 1.2% agarose gel, and the blot was subjected to hybridization. The arrows indicate precursors to 5.8S rRNA. The positions of mature rRNAs are indicated. Lower panel shows ethidium bromide staining of rRNAs in the same gel. (E) Pre-5.8S rRNA accumulated in cells depleted of Drosha, Ago2 or Dicer. Total RNA as used in (C) was separated in an 8% polyacrylamide, 7M urea gel and the blot was hybridized using the same probe as in (D). The arrows indicate different pre-5.8S rRNA species (marked as A, B and C). U3 snoRNA was probed to serve as a loading control. Lower panel shows ethidium bromide staining of rRNAs in the same gel.

Mentions: In eukaryotes, 18S, 5.8S and 28S rRNAs are transcribed by RNA polymerase I into a polycistronic molecule. This precursor is sequentially processed in the nucleolus (and nucleus) by multiple steps of endonucleolytic cleavage and exonucleolytic trimming reactions to produce mature rRNAs (8–10). In vertebrates, the longest detectable transcript, a 47S pre-rRNA containing the three rRNAs, 5′ and 3′ external transcribed spacers (ETS), and two internal transcribed spacers (ITS1 and ITS2), is processed by two alternative pathways to separate small and large subunit rRNAs (Figure 1A).Figure 1.


Depletion of key protein components of the RISC pathway impairs pre-ribosomal RNA processing.

Liang XH, Crooke ST - Nucleic Acids Res. (2011)

Pre-rRNA accumulation in cells depleted of RISC pathway proteins. (A) Pre-rRNA processing pathway in mammals. ETS and ITS are external and internal transcribed spacers, respectively. The position of the hybridization probe used in (D and E) is shown as a solid bar above 47S pre-rRNA. (B) mRNA levels were dramatically reduced 48 h after treatment with 50 nM ASOs targeting Drosha (ISIS25690), Ago2 (ISIS136764) or Dicer (ISIS138648), as determined by qRT–PCR. The error bars indicate standard deviation from two independent experiments with three replicates. UTC, untreated cells; +ASO, cells treated with ASOs. (C) The levels of targeted proteins were significantly reduced by ASO treatment, as determined by western analysis. Alpha-tubulin was used as a control for loading. (D) Northern hybridization for pre-rRNA species using a probe specific to the boundary of 5.8S rRNA/ITS2. Total RNA prepared from test cells 48 h after ASO treatment was separated on a 1.2% agarose gel, and the blot was subjected to hybridization. The arrows indicate precursors to 5.8S rRNA. The positions of mature rRNAs are indicated. Lower panel shows ethidium bromide staining of rRNAs in the same gel. (E) Pre-5.8S rRNA accumulated in cells depleted of Drosha, Ago2 or Dicer. Total RNA as used in (C) was separated in an 8% polyacrylamide, 7M urea gel and the blot was hybridized using the same probe as in (D). The arrows indicate different pre-5.8S rRNA species (marked as A, B and C). U3 snoRNA was probed to serve as a loading control. Lower panel shows ethidium bromide staining of rRNAs in the same gel.
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Figure 1: Pre-rRNA accumulation in cells depleted of RISC pathway proteins. (A) Pre-rRNA processing pathway in mammals. ETS and ITS are external and internal transcribed spacers, respectively. The position of the hybridization probe used in (D and E) is shown as a solid bar above 47S pre-rRNA. (B) mRNA levels were dramatically reduced 48 h after treatment with 50 nM ASOs targeting Drosha (ISIS25690), Ago2 (ISIS136764) or Dicer (ISIS138648), as determined by qRT–PCR. The error bars indicate standard deviation from two independent experiments with three replicates. UTC, untreated cells; +ASO, cells treated with ASOs. (C) The levels of targeted proteins were significantly reduced by ASO treatment, as determined by western analysis. Alpha-tubulin was used as a control for loading. (D) Northern hybridization for pre-rRNA species using a probe specific to the boundary of 5.8S rRNA/ITS2. Total RNA prepared from test cells 48 h after ASO treatment was separated on a 1.2% agarose gel, and the blot was subjected to hybridization. The arrows indicate precursors to 5.8S rRNA. The positions of mature rRNAs are indicated. Lower panel shows ethidium bromide staining of rRNAs in the same gel. (E) Pre-5.8S rRNA accumulated in cells depleted of Drosha, Ago2 or Dicer. Total RNA as used in (C) was separated in an 8% polyacrylamide, 7M urea gel and the blot was hybridized using the same probe as in (D). The arrows indicate different pre-5.8S rRNA species (marked as A, B and C). U3 snoRNA was probed to serve as a loading control. Lower panel shows ethidium bromide staining of rRNAs in the same gel.
Mentions: In eukaryotes, 18S, 5.8S and 28S rRNAs are transcribed by RNA polymerase I into a polycistronic molecule. This precursor is sequentially processed in the nucleolus (and nucleus) by multiple steps of endonucleolytic cleavage and exonucleolytic trimming reactions to produce mature rRNAs (8–10). In vertebrates, the longest detectable transcript, a 47S pre-rRNA containing the three rRNAs, 5′ and 3′ external transcribed spacers (ETS), and two internal transcribed spacers (ITS1 and ITS2), is processed by two alternative pathways to separate small and large subunit rRNAs (Figure 1A).Figure 1.

Bottom Line: Here, we show that depletion of key proteins of the RISC pathway by antisense oligonucleotides significantly impairs pre-rRNA processing in human cells.Both Dicer and Ago2 were detected in the nuclear fraction, and reduction of Dicer altered the structure of the nucleolus, where pre-rRNA processing occurs.Together, these results suggest that Drosha and Dicer are implicated in rRNA biogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Core Antisense Research, ISIS Pharmaceuticals, Inc., 1896 Rutherford Rd, Carlsbad, CA 92008, USA. lliang@isisph.com

ABSTRACT
Little is known about whether components of the RNA-induced silencing complex (RISC) mediate the biogenesis of RNAs other than miRNA. Here, we show that depletion of key proteins of the RISC pathway by antisense oligonucleotides significantly impairs pre-rRNA processing in human cells. In cells depleted of Drosha or Dicer, different precursors to 5.8S rRNA strongly accumulated, without affecting normal endonucleolytic cleavages. Moderate yet distinct processing defects were also observed in Ago2-depleted cells. Physical links between pre-rRNA and these proteins were identified by co-immunoprecipitation analyses. Interestingly, simultaneous depletion of Dicer and Drosha led to a different processing defect, causing slower production of 28S rRNA and its precursor. Both Dicer and Ago2 were detected in the nuclear fraction, and reduction of Dicer altered the structure of the nucleolus, where pre-rRNA processing occurs. Together, these results suggest that Drosha and Dicer are implicated in rRNA biogenesis.

Show MeSH