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miRISC recruits decapping factors to miRNA targets to enhance their degradation.

Nishihara T, Zekri L, Braun JE, Izaurralde E - Nucleic Acids Res. (2013)

Bottom Line: Remarkably, miRISC recruits DCP1, Me31B and HPat to engineered miRNA targets transcribed by RNA polymerase III, which lack a cap structure, a protein-coding region and a poly(A) tail.Furthermore, miRISC can trigger decapping and the subsequent degradation of mRNA targets independently of ongoing deadenylation.Thus, miRISC increases the local concentration of the decapping machinery on miRNA targets to facilitate decapping and irreversibly shut down their translation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Max Planck Institute for Developmental Biology, Spemannstrasse 35, 72076 Tübingen, Germany.

ABSTRACT
MicroRNA (miRNA)-induced silencing complexes (miRISCs) repress translation and promote degradation of miRNA targets. Target degradation occurs through the 5'-to-3' messenger RNA (mRNA) decay pathway, wherein, after shortening of the mRNA poly(A) tail, the removal of the 5' cap structure by decapping triggers irreversible decay of the mRNA body. Here, we demonstrate that miRISC enhances the association of the decapping activators DCP1, Me31B and HPat with deadenylated miRNA targets that accumulate when decapping is blocked. DCP1 and Me31B recruitment by miRISC occurs before the completion of deadenylation. Remarkably, miRISC recruits DCP1, Me31B and HPat to engineered miRNA targets transcribed by RNA polymerase III, which lack a cap structure, a protein-coding region and a poly(A) tail. Furthermore, miRISC can trigger decapping and the subsequent degradation of mRNA targets independently of ongoing deadenylation. Thus, miRISC increases the local concentration of the decapping machinery on miRNA targets to facilitate decapping and irreversibly shut down their translation.

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miRNAs enhance the association of Me31B, HPat and DCP1 with miRNA targets. (A) The association of GST-tagged AGO1 with the F-Luc-CG5281 mRNA reporter was analyzed in the absence or presence of miR-12 in cells expressing the DCP2* catalytic mutant. GST-tagged MBP (maltose-binding protein) served as negative control. The pull downs were performed in cells expressing V5-GW182 or the corresponding empty vector (−). RNAs in the input and pull-down fractions were analyzed by northern blotting. Ribosomal RNA (rRNA) and a spike RNA were visualized by ethidium bromide staining. (B) The association of V5-tagged GW182 [wild-type (WT) or the 12xGW mutant] with the F-Luc-CG5281 mRNA reporter was analyzed in the absence or presence of miR-12. V5-MBP served as a negative control. RNAs in the inputs and immunoprecipitates (IP) were analyzed as described in panel (A). (C–E) The association of GST-tagged Me31B and HPat or of HA-tagged DCP1 with the F-Luc-CG5281 reporter in the absence (−) or presence (+) of miR-12 was analyzed as described in panel (A). HA-DCP1 was immunoprecipitated using anti-HA antibodies. HA-MBP served as a negative control. The DCP2* mutant was included in panels (B–D) but not in panel (E) because DCP1 overexpression inhibits decapping of the miRNA target as is evident by the accumulation of the deadenylated F-Luc-CG5281 mRNA in the presence of miR-12 (Figure 1E, input, lanes 2 and 4 versus 1 and 3). In this panel, untagged DCP1 was included in cells expressing HA-MBP to prevent degradation of the reporter. The position of the deadenylated (A0) mRNA reporter is indicated.
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gkt619-F1: miRNAs enhance the association of Me31B, HPat and DCP1 with miRNA targets. (A) The association of GST-tagged AGO1 with the F-Luc-CG5281 mRNA reporter was analyzed in the absence or presence of miR-12 in cells expressing the DCP2* catalytic mutant. GST-tagged MBP (maltose-binding protein) served as negative control. The pull downs were performed in cells expressing V5-GW182 or the corresponding empty vector (−). RNAs in the input and pull-down fractions were analyzed by northern blotting. Ribosomal RNA (rRNA) and a spike RNA were visualized by ethidium bromide staining. (B) The association of V5-tagged GW182 [wild-type (WT) or the 12xGW mutant] with the F-Luc-CG5281 mRNA reporter was analyzed in the absence or presence of miR-12. V5-MBP served as a negative control. RNAs in the inputs and immunoprecipitates (IP) were analyzed as described in panel (A). (C–E) The association of GST-tagged Me31B and HPat or of HA-tagged DCP1 with the F-Luc-CG5281 reporter in the absence (−) or presence (+) of miR-12 was analyzed as described in panel (A). HA-DCP1 was immunoprecipitated using anti-HA antibodies. HA-MBP served as a negative control. The DCP2* mutant was included in panels (B–D) but not in panel (E) because DCP1 overexpression inhibits decapping of the miRNA target as is evident by the accumulation of the deadenylated F-Luc-CG5281 mRNA in the presence of miR-12 (Figure 1E, input, lanes 2 and 4 versus 1 and 3). In this panel, untagged DCP1 was included in cells expressing HA-MBP to prevent degradation of the reporter. The position of the deadenylated (A0) mRNA reporter is indicated.

Mentions: Because the mRNA target is partially degraded at the steady state (Supplementary Figure S2A; e.g. lane 2 versus 1), to avoid target degradation and trap mRNA decay intermediates, the pull-down experiments were repeated in S2 cells overexpressing a catalytically inactive DCP2 mutant (DCP2*, carrying the E361A substitution). In these cells, miRNA targets accumulate in a deadenylated form (because deadenylation precedes decapping), but decapping and further degradation are partially inhibited (Figure 1A versus Supplementary Figure S2A) (15–18). We observed that GST-AGO1 pulled down the deadenylated reporter in a miR-12-dependent manner (Figure 1A, lanes 4 and 8). Western blot analyses indicated that GST-AGO1 was expressed and pulled down at comparable levels in the presence or absence of miR-12 (Supplementary Figure S2B).Figure 1.


miRISC recruits decapping factors to miRNA targets to enhance their degradation.

Nishihara T, Zekri L, Braun JE, Izaurralde E - Nucleic Acids Res. (2013)

miRNAs enhance the association of Me31B, HPat and DCP1 with miRNA targets. (A) The association of GST-tagged AGO1 with the F-Luc-CG5281 mRNA reporter was analyzed in the absence or presence of miR-12 in cells expressing the DCP2* catalytic mutant. GST-tagged MBP (maltose-binding protein) served as negative control. The pull downs were performed in cells expressing V5-GW182 or the corresponding empty vector (−). RNAs in the input and pull-down fractions were analyzed by northern blotting. Ribosomal RNA (rRNA) and a spike RNA were visualized by ethidium bromide staining. (B) The association of V5-tagged GW182 [wild-type (WT) or the 12xGW mutant] with the F-Luc-CG5281 mRNA reporter was analyzed in the absence or presence of miR-12. V5-MBP served as a negative control. RNAs in the inputs and immunoprecipitates (IP) were analyzed as described in panel (A). (C–E) The association of GST-tagged Me31B and HPat or of HA-tagged DCP1 with the F-Luc-CG5281 reporter in the absence (−) or presence (+) of miR-12 was analyzed as described in panel (A). HA-DCP1 was immunoprecipitated using anti-HA antibodies. HA-MBP served as a negative control. The DCP2* mutant was included in panels (B–D) but not in panel (E) because DCP1 overexpression inhibits decapping of the miRNA target as is evident by the accumulation of the deadenylated F-Luc-CG5281 mRNA in the presence of miR-12 (Figure 1E, input, lanes 2 and 4 versus 1 and 3). In this panel, untagged DCP1 was included in cells expressing HA-MBP to prevent degradation of the reporter. The position of the deadenylated (A0) mRNA reporter is indicated.
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Related In: Results  -  Collection

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gkt619-F1: miRNAs enhance the association of Me31B, HPat and DCP1 with miRNA targets. (A) The association of GST-tagged AGO1 with the F-Luc-CG5281 mRNA reporter was analyzed in the absence or presence of miR-12 in cells expressing the DCP2* catalytic mutant. GST-tagged MBP (maltose-binding protein) served as negative control. The pull downs were performed in cells expressing V5-GW182 or the corresponding empty vector (−). RNAs in the input and pull-down fractions were analyzed by northern blotting. Ribosomal RNA (rRNA) and a spike RNA were visualized by ethidium bromide staining. (B) The association of V5-tagged GW182 [wild-type (WT) or the 12xGW mutant] with the F-Luc-CG5281 mRNA reporter was analyzed in the absence or presence of miR-12. V5-MBP served as a negative control. RNAs in the inputs and immunoprecipitates (IP) were analyzed as described in panel (A). (C–E) The association of GST-tagged Me31B and HPat or of HA-tagged DCP1 with the F-Luc-CG5281 reporter in the absence (−) or presence (+) of miR-12 was analyzed as described in panel (A). HA-DCP1 was immunoprecipitated using anti-HA antibodies. HA-MBP served as a negative control. The DCP2* mutant was included in panels (B–D) but not in panel (E) because DCP1 overexpression inhibits decapping of the miRNA target as is evident by the accumulation of the deadenylated F-Luc-CG5281 mRNA in the presence of miR-12 (Figure 1E, input, lanes 2 and 4 versus 1 and 3). In this panel, untagged DCP1 was included in cells expressing HA-MBP to prevent degradation of the reporter. The position of the deadenylated (A0) mRNA reporter is indicated.
Mentions: Because the mRNA target is partially degraded at the steady state (Supplementary Figure S2A; e.g. lane 2 versus 1), to avoid target degradation and trap mRNA decay intermediates, the pull-down experiments were repeated in S2 cells overexpressing a catalytically inactive DCP2 mutant (DCP2*, carrying the E361A substitution). In these cells, miRNA targets accumulate in a deadenylated form (because deadenylation precedes decapping), but decapping and further degradation are partially inhibited (Figure 1A versus Supplementary Figure S2A) (15–18). We observed that GST-AGO1 pulled down the deadenylated reporter in a miR-12-dependent manner (Figure 1A, lanes 4 and 8). Western blot analyses indicated that GST-AGO1 was expressed and pulled down at comparable levels in the presence or absence of miR-12 (Supplementary Figure S2B).Figure 1.

Bottom Line: Remarkably, miRISC recruits DCP1, Me31B and HPat to engineered miRNA targets transcribed by RNA polymerase III, which lack a cap structure, a protein-coding region and a poly(A) tail.Furthermore, miRISC can trigger decapping and the subsequent degradation of mRNA targets independently of ongoing deadenylation.Thus, miRISC increases the local concentration of the decapping machinery on miRNA targets to facilitate decapping and irreversibly shut down their translation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Max Planck Institute for Developmental Biology, Spemannstrasse 35, 72076 Tübingen, Germany.

ABSTRACT
MicroRNA (miRNA)-induced silencing complexes (miRISCs) repress translation and promote degradation of miRNA targets. Target degradation occurs through the 5'-to-3' messenger RNA (mRNA) decay pathway, wherein, after shortening of the mRNA poly(A) tail, the removal of the 5' cap structure by decapping triggers irreversible decay of the mRNA body. Here, we demonstrate that miRISC enhances the association of the decapping activators DCP1, Me31B and HPat with deadenylated miRNA targets that accumulate when decapping is blocked. DCP1 and Me31B recruitment by miRISC occurs before the completion of deadenylation. Remarkably, miRISC recruits DCP1, Me31B and HPat to engineered miRNA targets transcribed by RNA polymerase III, which lack a cap structure, a protein-coding region and a poly(A) tail. Furthermore, miRISC can trigger decapping and the subsequent degradation of mRNA targets independently of ongoing deadenylation. Thus, miRISC increases the local concentration of the decapping machinery on miRNA targets to facilitate decapping and irreversibly shut down their translation.

Show MeSH
Related in: MedlinePlus