miRISC recruits decapping factors to miRNA targets to enhance their degradation

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.

Figure 1.

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.

Figure 2.

AGO1 and GW182 associate with miRNA targets lacking a 5′ cap structure and a poly(A) tail. (A) Schematic representation of Dm 7SL RNA and the Alu-miRNA targets that we termed EvAluators. (B) The association of the Alu-miR-12 RNA with HA-tagged MBP or eIF4E in control cells or in cells expressing V5-GW182 was analyzed by co-immunoprecipitation using anti-HA antibodies. RNA samples were analyzed as described in Figure 1A. (C) The association of endogenous PABPC1 with the Alu-miR-12 RNA was analyzed using a polyclonal anti-PABPC1 antibody. The corresponding preimmune (Pre) serum served as a negative control. (D and E) The association of the Alu-miR-12 target with GST-AGO1 and V5-GW182 (wild-type or 12xGW mutant) in the absence or presence of miR-12 was analyzed as described in Figure 1.

Figure 3.

DCP1, HPat and Me31B are recruited to the Alu-miRNA targets. (A) The association of HA-DCP1 with the Alu-miR-12 target was analyzed as described in Figure 1, except that DCP2* was omitted. (B) The specificity of the association of DCP1 with the Alu-miR-12 or Alu-miR-1 target was analyzed in the presence of the corresponding miRNAs. (C) The association of GST-HPat and GST-Me31B with the Alu-miR-12 target was analyzed as described in Figure 1. (D) Association of GST-Me31B with the Alu-miR-1 target in the presence of miR-1 or miR-12.

Figure 4.

DCP1 and Me31B associate with miRNA targets independently of the length of the poly(A) tail. (A–C) The association of GST-tagged MBP, HPat, Me31B, AGO1 and GW182 with F-Luc-CG5281 in the absence or presence of miR-12 was analyzed in cells overexpressing the DCP2* or POP2* mutant as described in Figure 1. (B and C) The association of HA-DCP1 with a miR-12 or miR-1 target was analyzed in cells expressing the DCP2* or POP2* mutant. The positions of the polyadenylated (A₁₄₀, dotted line) and deadenylated (A₀) mRNA are indicated in all panels.

Figure 5.

miRISC induces deadenylation-independent decapping. (A) Schematic representation of the F-Luc-miRNA-5BoxB-HhR reporter containing miR-12 or miR-1-binding sites, five BoxB hairpins and a self-cleavable HhR. (B and C) The graphs represent F-Luc activities (black bars) and mRNA levels (gray bars) normalized to those of Renilla luciferase in the absence or presence of λN-PABPC1 in control cells in the absence of miRNAs. The values were set to 1 in the absence of λN-PABPC1. (D and E) The graphs represent F-Luc activities (black bars) and mRNA levels (gray bars) normalized to those of Renilla luciferase in the absence or presence of miRNAs in control cells or in cells depleted of DCP2 expressing a dsRNA-resistant version of DCP2 (wild-type, WT or the catalytic DCP2* mutant). For each condition, the normalized values of F-Luc activity and mRNA levels were set at 100 in the absence of the miRNA. The mean values ± standard deviations from three independent experiments are shown in all panels. Dashed lines indicate F-Luc activity (black) and mRNA levels (gray) in control cells expressing the miRNA. (F and G) Northern blots of representative RNA samples corresponding to the graphs shown in panels (C) and (F), respectively.

Figure 6.

Schematic representation of the F-Luc-miR-12-(A)₉₃-HhR reporter. The F-Luc-miR-12-(A)₉₃-HhR reporter contains three miR-12-binding sites, consecutive poly(A) and poly(C) stretches of 93 and 6 residues, respectively, and a self-cleavable HhR. (B–D) S2 cells were transfected with a mixture of three plasmids: one expressing the indicated F-Luc reporters, another expressing the miR-12 primary transcript or the corresponding empty vector (-miR-12) and a third expressing Renilla luciferase (R-Luc). The transfection mixtures contained plasmids expressing GST-XRN1 or GST as a negative control. F-Luc activity (black bars) and mRNA levels (gray bars) were normalized to those of the Renilla luciferase and set at 100 in the absence of miR-12. The mean values ± standard deviations from three independent experiments are shown in panels (B and C). (D) Northern blot of representative RNA samples.