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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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Cells lacking Dicer exhibit altered nucleolar structure. Indirect immunofluorescence was performed for cells depleted of Dicer, Drosha or Ago2 using first antibody against nucleolin and secondary anti-rabbit antibody conjugated to Texas Red (red). DNA was stained with DAPI (blue). Arrows indicate aggregated nucleoli in cells depleted of Dicer.
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Figure 5: Cells lacking Dicer exhibit altered nucleolar structure. Indirect immunofluorescence was performed for cells depleted of Dicer, Drosha or Ago2 using first antibody against nucleolin and secondary anti-rabbit antibody conjugated to Texas Red (red). DNA was stained with DAPI (blue). Arrows indicate aggregated nucleoli in cells depleted of Dicer.

Mentions: In eukaryotes, pre-rRNA is transcribed and processed in the nucleolus, which is a tripartite structure in terms of known functions. Pre-rRNA is transcribed in the fibrillar center (FC), and initially processed in a surrounding domain known as dense fibrillar center (DFC). Late processing events occur in a third structure, the granular component (GC) (23). Certain step(s) of pre-rRNA processing also occurs in the nucleoplasm (24). Since loss of Drosha or Dicer affects rRNA processing, we reasoned that nucleolar structure might be affected as well. Thus, indirect immunofluorescence was performed to stain a nucleolar marker protein, Nucleolin, which localizes in DFC and GC. This protein is also known to be required for pre-rRNA processing (25). Significant difference in the nucleolar structure was found in cells depleted of Dicer, which exhibit fewer, but larger, aggregated nucleoli than control cells (Figure 5). This difference is significant, since ∼65% of cells treated with the Dicer ASO contained aggregated nucleoli, whereas <10% of control cells showed similar nucleolar structures (data not shown). No significant difference in nucleolar structure was observed in cells depleted of Ago2 or Drosha, although reduction of Drosha caused processing defects similar to depletion of Dicer. Together, these results indicate that reduction of Dicer alters nucleolar structure, directly or indirectly, and that Dicer and Drosha may affect pre-rRNA processing in different ways.Figure 5.


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

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

Cells lacking Dicer exhibit altered nucleolar structure. Indirect immunofluorescence was performed for cells depleted of Dicer, Drosha or Ago2 using first antibody against nucleolin and secondary anti-rabbit antibody conjugated to Texas Red (red). DNA was stained with DAPI (blue). Arrows indicate aggregated nucleoli in cells depleted of Dicer.
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Related In: Results  -  Collection

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Figure 5: Cells lacking Dicer exhibit altered nucleolar structure. Indirect immunofluorescence was performed for cells depleted of Dicer, Drosha or Ago2 using first antibody against nucleolin and secondary anti-rabbit antibody conjugated to Texas Red (red). DNA was stained with DAPI (blue). Arrows indicate aggregated nucleoli in cells depleted of Dicer.
Mentions: In eukaryotes, pre-rRNA is transcribed and processed in the nucleolus, which is a tripartite structure in terms of known functions. Pre-rRNA is transcribed in the fibrillar center (FC), and initially processed in a surrounding domain known as dense fibrillar center (DFC). Late processing events occur in a third structure, the granular component (GC) (23). Certain step(s) of pre-rRNA processing also occurs in the nucleoplasm (24). Since loss of Drosha or Dicer affects rRNA processing, we reasoned that nucleolar structure might be affected as well. Thus, indirect immunofluorescence was performed to stain a nucleolar marker protein, Nucleolin, which localizes in DFC and GC. This protein is also known to be required for pre-rRNA processing (25). Significant difference in the nucleolar structure was found in cells depleted of Dicer, which exhibit fewer, but larger, aggregated nucleoli than control cells (Figure 5). This difference is significant, since ∼65% of cells treated with the Dicer ASO contained aggregated nucleoli, whereas <10% of control cells showed similar nucleolar structures (data not shown). No significant difference in nucleolar structure was observed in cells depleted of Ago2 or Drosha, although reduction of Drosha caused processing defects similar to depletion of Dicer. Together, these results indicate that reduction of Dicer alters nucleolar structure, directly or indirectly, and that Dicer and Drosha may affect pre-rRNA processing in different ways.Figure 5.

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