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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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Loss of RISC proteins leads to accumulation of different pre-5.8S rRNA species. (A) Depiction of probe positions around the 5.8S rRNA region. The probe names are shown under the pre-rRNA. (B) Total RNA was prepared from test cells 48 h after ASO treatment, and subjected to northern hybridization. The same membrane was hybridized sequentially using probes specific to 5′ (left panel, 5′ probe), 5.8S (middle panel, 5.8S probe) and 3′ regions (right panel, 3′ probe), respectively. U3 snoRNA was used as a loading control. Different pre-5.8S rRNA species are indicated. The asterisks indicate RNA species that were only detected with one probe. M, size maker in nucleotides (low range ssRNA ladder, Biolabs); 5.8S-L, the longer version of mature 5.8S rRNA; shorter exposure for 5.8S-L rRNA is shown in left panel, lower part. (C) Summary of changes in the levels of different pre-5.8S rRNA species in cells depleted of the RISC pathway proteins. App. size, estimated nucleotide length of the RNA. Extension, types of 5′ or 3′ flanking sequences in pre-5.8S rRNAs. The larger arrows indicate stronger accumulation.
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Figure 2: Loss of RISC proteins leads to accumulation of different pre-5.8S rRNA species. (A) Depiction of probe positions around the 5.8S rRNA region. The probe names are shown under the pre-rRNA. (B) Total RNA was prepared from test cells 48 h after ASO treatment, and subjected to northern hybridization. The same membrane was hybridized sequentially using probes specific to 5′ (left panel, 5′ probe), 5.8S (middle panel, 5.8S probe) and 3′ regions (right panel, 3′ probe), respectively. U3 snoRNA was used as a loading control. Different pre-5.8S rRNA species are indicated. The asterisks indicate RNA species that were only detected with one probe. M, size maker in nucleotides (low range ssRNA ladder, Biolabs); 5.8S-L, the longer version of mature 5.8S rRNA; shorter exposure for 5.8S-L rRNA is shown in left panel, lower part. (C) Summary of changes in the levels of different pre-5.8S rRNA species in cells depleted of the RISC pathway proteins. App. size, estimated nucleotide length of the RNA. Extension, types of 5′ or 3′ flanking sequences in pre-5.8S rRNAs. The larger arrows indicate stronger accumulation.

Mentions: All the pre-5.8S rRNA species detected in Figure 1 contain 3′ extensions, since the hybridization probe recognizes only this subset of pre-rRNAs. The two major bands (∼300 and ∼400 nt) are identical in size to the two previously reported precursors, which contain mature 5′-end of 5.8S rRNA and 3′ extensions with ∼156 or ∼250 nt ITS2 sequence, respectively (13,22). These data suggest that the two precursors we detected here also contain only the 3′ extensions. To confirm this, and to determine if loss of these proteins leads to accumulation of precursors containing 5′ extension, high resolution northern hybridization was performed using probes specific to 5.8S rRNA (5.8S), or the 5′, or the 3′ boundaries of the 5.8S rRNA (Figure 2A). The 5′ or 3′ probes recognize precursors containing ITS1 sequence at 5′-end of 5.8S rRNA, or ITS2 sequence at 3′-end of 5.8S rRNA, respectively. However, the 5.8S probe can detect precursors containing either 5′ or 3′ extension, or both.Figure 2.


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

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

Loss of RISC proteins leads to accumulation of different pre-5.8S rRNA species. (A) Depiction of probe positions around the 5.8S rRNA region. The probe names are shown under the pre-rRNA. (B) Total RNA was prepared from test cells 48 h after ASO treatment, and subjected to northern hybridization. The same membrane was hybridized sequentially using probes specific to 5′ (left panel, 5′ probe), 5.8S (middle panel, 5.8S probe) and 3′ regions (right panel, 3′ probe), respectively. U3 snoRNA was used as a loading control. Different pre-5.8S rRNA species are indicated. The asterisks indicate RNA species that were only detected with one probe. M, size maker in nucleotides (low range ssRNA ladder, Biolabs); 5.8S-L, the longer version of mature 5.8S rRNA; shorter exposure for 5.8S-L rRNA is shown in left panel, lower part. (C) Summary of changes in the levels of different pre-5.8S rRNA species in cells depleted of the RISC pathway proteins. App. size, estimated nucleotide length of the RNA. Extension, types of 5′ or 3′ flanking sequences in pre-5.8S rRNAs. The larger arrows indicate stronger accumulation.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 2: Loss of RISC proteins leads to accumulation of different pre-5.8S rRNA species. (A) Depiction of probe positions around the 5.8S rRNA region. The probe names are shown under the pre-rRNA. (B) Total RNA was prepared from test cells 48 h after ASO treatment, and subjected to northern hybridization. The same membrane was hybridized sequentially using probes specific to 5′ (left panel, 5′ probe), 5.8S (middle panel, 5.8S probe) and 3′ regions (right panel, 3′ probe), respectively. U3 snoRNA was used as a loading control. Different pre-5.8S rRNA species are indicated. The asterisks indicate RNA species that were only detected with one probe. M, size maker in nucleotides (low range ssRNA ladder, Biolabs); 5.8S-L, the longer version of mature 5.8S rRNA; shorter exposure for 5.8S-L rRNA is shown in left panel, lower part. (C) Summary of changes in the levels of different pre-5.8S rRNA species in cells depleted of the RISC pathway proteins. App. size, estimated nucleotide length of the RNA. Extension, types of 5′ or 3′ flanking sequences in pre-5.8S rRNAs. The larger arrows indicate stronger accumulation.
Mentions: All the pre-5.8S rRNA species detected in Figure 1 contain 3′ extensions, since the hybridization probe recognizes only this subset of pre-rRNAs. The two major bands (∼300 and ∼400 nt) are identical in size to the two previously reported precursors, which contain mature 5′-end of 5.8S rRNA and 3′ extensions with ∼156 or ∼250 nt ITS2 sequence, respectively (13,22). These data suggest that the two precursors we detected here also contain only the 3′ extensions. To confirm this, and to determine if loss of these proteins leads to accumulation of precursors containing 5′ extension, high resolution northern hybridization was performed using probes specific to 5.8S rRNA (5.8S), or the 5′, or the 3′ boundaries of the 5.8S rRNA (Figure 2A). The 5′ or 3′ probes recognize precursors containing ITS1 sequence at 5′-end of 5.8S rRNA, or ITS2 sequence at 3′-end of 5.8S rRNA, respectively. However, the 5.8S probe can detect precursors containing either 5′ or 3′ extension, or both.Figure 2.

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
Related in: MedlinePlus