Limits...
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.

Show MeSH
Pre-rRNA containing 5.8S rRNA and flanking ITS sequences can be co-immunoprecipitated with RISC proteins. Immunoprecipitation was carried out using antibodies against Drosha, Ago2 or Dicer. Co-selected RNAs were analyzed by RT–PCR. (A) The positions of probe sets in pre-rRNA used for RT–PCR reaction are indicated. The expected sizes of PCR product are given. Co-immunoprecipitated RNAs were subjected to reverse transcription with (+RT) or without (–RT) oligonucleotides complementary to different regions of pre-rRNA or tubulin mRNA. RT reactions were used as templates for PCR amplification. The PCR products were analyzed on 2% agarose gels. Input, RNA prepared from 10% of the material used for immunoprecipitation. –AB, control immunoprecipitation experiment without antibody; M, 1 kb plus DNA ladder. The sizes are shown in base pairs. (B and C) RT–PCR detection for pre-rRNAs containing 5.8S/ITS2 and ITS1/5.8S regions, respectively. (D) RT–PCR for 18S/ITS1 region (upper panel) or 28S/3′ETS region (lower panel). (E) RT–PCR for β-tubulin mRNA. (F) Immunoprecipitation was performed using an antibody against a splicing factor (SF3B3), and the precipitated RNA was subjected to RT–PCR for ITS1/5.8S (3′ITS1) or 5.8S/ITS2 (ITS2) regions.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3113584&req=5

Figure 6: Pre-rRNA containing 5.8S rRNA and flanking ITS sequences can be co-immunoprecipitated with RISC proteins. Immunoprecipitation was carried out using antibodies against Drosha, Ago2 or Dicer. Co-selected RNAs were analyzed by RT–PCR. (A) The positions of probe sets in pre-rRNA used for RT–PCR reaction are indicated. The expected sizes of PCR product are given. Co-immunoprecipitated RNAs were subjected to reverse transcription with (+RT) or without (–RT) oligonucleotides complementary to different regions of pre-rRNA or tubulin mRNA. RT reactions were used as templates for PCR amplification. The PCR products were analyzed on 2% agarose gels. Input, RNA prepared from 10% of the material used for immunoprecipitation. –AB, control immunoprecipitation experiment without antibody; M, 1 kb plus DNA ladder. The sizes are shown in base pairs. (B and C) RT–PCR detection for pre-rRNAs containing 5.8S/ITS2 and ITS1/5.8S regions, respectively. (D) RT–PCR for 18S/ITS1 region (upper panel) or 28S/3′ETS region (lower panel). (E) RT–PCR for β-tubulin mRNA. (F) Immunoprecipitation was performed using an antibody against a splicing factor (SF3B3), and the precipitated RNA was subjected to RT–PCR for ITS1/5.8S (3′ITS1) or 5.8S/ITS2 (ITS2) regions.

Mentions: Immunoprecipitation was carried out at 150 mM salt concentration using antibodies against Dicer, Drosha or Ago2. The co-selection of pre-rRNAs was examined by RT–PCR. Four sets of primers were used that are specific to different regions of pre-rRNA: the boundaries of 18S rRNA/ITS1 (5′ITS1), ITS1/5.8S rRNA (3′ITS1), 5.8S rRNA/ITS2 (ITS2) and 28S rRNA/3′ ETS (3′ETS) (Figure 6A). A single RT–PCR product specific to 5.8S/ITS2 primers was detected at the expected size with the precipitated RNAs using Drosha, Ago2 or Dicer antibodies (Figure 6B, upper panel, lanes 3–5), but no signal was detected in control experiments without antibody. Similarly, an RT–PCR product specific to the junction of ITS1/5.8S rRNA was also detected using 3′ITS1 primers (Figure 6C). The relative recovery of the co-precipitated pre-rRNA was ∼1–3% of total, as estimated based on the PCR signal detected with the RNA sample prepared from 10% of whole-cell extract used in immunoprecipitation experiment, suggesting that only a fraction of pre-rRNA is linked with these proteins. However, neither pre-rRNA(s) containing 18S/ITS1 or 3′ ETS sequence nor β-tubulin mRNA was detected, as determined using primers specific to 5′ITS1, 3′ETS or tubulin mRNA, respectively (Figure 6D and E). Importantly, the pre-rRNA(s) co-selected with the RISC proteins were not co-precipitated with an antibody against splicing factor SF3B3 (Figure 6F), suggesting that pre-rRNA could be specifically precipitated with the RISC pathway proteins. Together, the results suggest that these proteins are physically linked to pre-rRNA species containing the 5.8S rRNA and flanking sequences, either directly or mediated by other components. In addition, these data also strongly argue that the defect on pre-rRNA processing is a direct effect caused by loss of the RISC pathway proteins.Figure 6.


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

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

Pre-rRNA containing 5.8S rRNA and flanking ITS sequences can be co-immunoprecipitated with RISC proteins. Immunoprecipitation was carried out using antibodies against Drosha, Ago2 or Dicer. Co-selected RNAs were analyzed by RT–PCR. (A) The positions of probe sets in pre-rRNA used for RT–PCR reaction are indicated. The expected sizes of PCR product are given. Co-immunoprecipitated RNAs were subjected to reverse transcription with (+RT) or without (–RT) oligonucleotides complementary to different regions of pre-rRNA or tubulin mRNA. RT reactions were used as templates for PCR amplification. The PCR products were analyzed on 2% agarose gels. Input, RNA prepared from 10% of the material used for immunoprecipitation. –AB, control immunoprecipitation experiment without antibody; M, 1 kb plus DNA ladder. The sizes are shown in base pairs. (B and C) RT–PCR detection for pre-rRNAs containing 5.8S/ITS2 and ITS1/5.8S regions, respectively. (D) RT–PCR for 18S/ITS1 region (upper panel) or 28S/3′ETS region (lower panel). (E) RT–PCR for β-tubulin mRNA. (F) Immunoprecipitation was performed using an antibody against a splicing factor (SF3B3), and the precipitated RNA was subjected to RT–PCR for ITS1/5.8S (3′ITS1) or 5.8S/ITS2 (ITS2) regions.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3113584&req=5

Figure 6: Pre-rRNA containing 5.8S rRNA and flanking ITS sequences can be co-immunoprecipitated with RISC proteins. Immunoprecipitation was carried out using antibodies against Drosha, Ago2 or Dicer. Co-selected RNAs were analyzed by RT–PCR. (A) The positions of probe sets in pre-rRNA used for RT–PCR reaction are indicated. The expected sizes of PCR product are given. Co-immunoprecipitated RNAs were subjected to reverse transcription with (+RT) or without (–RT) oligonucleotides complementary to different regions of pre-rRNA or tubulin mRNA. RT reactions were used as templates for PCR amplification. The PCR products were analyzed on 2% agarose gels. Input, RNA prepared from 10% of the material used for immunoprecipitation. –AB, control immunoprecipitation experiment without antibody; M, 1 kb plus DNA ladder. The sizes are shown in base pairs. (B and C) RT–PCR detection for pre-rRNAs containing 5.8S/ITS2 and ITS1/5.8S regions, respectively. (D) RT–PCR for 18S/ITS1 region (upper panel) or 28S/3′ETS region (lower panel). (E) RT–PCR for β-tubulin mRNA. (F) Immunoprecipitation was performed using an antibody against a splicing factor (SF3B3), and the precipitated RNA was subjected to RT–PCR for ITS1/5.8S (3′ITS1) or 5.8S/ITS2 (ITS2) regions.
Mentions: Immunoprecipitation was carried out at 150 mM salt concentration using antibodies against Dicer, Drosha or Ago2. The co-selection of pre-rRNAs was examined by RT–PCR. Four sets of primers were used that are specific to different regions of pre-rRNA: the boundaries of 18S rRNA/ITS1 (5′ITS1), ITS1/5.8S rRNA (3′ITS1), 5.8S rRNA/ITS2 (ITS2) and 28S rRNA/3′ ETS (3′ETS) (Figure 6A). A single RT–PCR product specific to 5.8S/ITS2 primers was detected at the expected size with the precipitated RNAs using Drosha, Ago2 or Dicer antibodies (Figure 6B, upper panel, lanes 3–5), but no signal was detected in control experiments without antibody. Similarly, an RT–PCR product specific to the junction of ITS1/5.8S rRNA was also detected using 3′ITS1 primers (Figure 6C). The relative recovery of the co-precipitated pre-rRNA was ∼1–3% of total, as estimated based on the PCR signal detected with the RNA sample prepared from 10% of whole-cell extract used in immunoprecipitation experiment, suggesting that only a fraction of pre-rRNA is linked with these proteins. However, neither pre-rRNA(s) containing 18S/ITS1 or 3′ ETS sequence nor β-tubulin mRNA was detected, as determined using primers specific to 5′ITS1, 3′ETS or tubulin mRNA, respectively (Figure 6D and E). Importantly, the pre-rRNA(s) co-selected with the RISC proteins were not co-precipitated with an antibody against splicing factor SF3B3 (Figure 6F), suggesting that pre-rRNA could be specifically precipitated with the RISC pathway proteins. Together, the results suggest that these proteins are physically linked to pre-rRNA species containing the 5.8S rRNA and flanking sequences, either directly or mediated by other components. In addition, these data also strongly argue that the defect on pre-rRNA processing is a direct effect caused by loss of the RISC pathway proteins.Figure 6.

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