MicroRNA-dependent localization of targeted mRNAs to mammalian P-bodies

Abstract

Small RNAs, including small interfering RNAs (siRNAs) and microRNAs (miRNAs) can silence target genes through several different effector mechanisms. Whereas siRNA-directed mRNA cleavage is increasingly understood, the mechanisms by which miRNAs repress protein synthesis are obscure. Recent studies have revealed the existence of specific cytoplasmic foci, referred to herein as processing bodies (P-bodies), which contain untranslated mRNAs and can serve as sites of mRNA degradation. Here we demonstrate that Argonaute proteins--the signature components of the RNA interference (RNAi) effector complex, RISC--localize to mammalian P-bodies. Moreover, reporter mRNAs that are targeted for translational repression by endogenous or exogenous miRNAs become concentrated in P-bodies in a miRNA-dependent manner. These results provide a link between miRNA function and mammalian P-bodies and suggest that translation repression by RISC delivers mRNAs to P-bodies, either as a cause or as a consequence of inhibiting protein synthesis.

Figure 1

Argonaute proteins localize to mammalian P-bodies. (a) Myc-tagged Ago2 protein was expressed in U2-OS cells. Ago2 protein localized to discrete cytoplasmic foci by staining with FITC-conjugated anti-Myc. (b) Endogenous Ago2 protein was localized in U2-OS cells by staining with a rabbit anti-Ago2 antibody. (c) Argonaute proteins colocalized with either GFP- or Flag-tagged Dcp1a, a signature component of the mammalian P-bodies. Argonaute proteins were visualized using a Rhodamine-Red-conjugated anti-Myc. Dcp1a was visualized either by GFP or FITC-conjugated anti-Flag.

Figure 2

Argonaute proteins bind components of mammalian P-bodies. Human 293T cells were transfected with Myc-tagged Ago1 or Ago2 in combination with either GFP-tagged Dcp1a or Flag-tagged Dcp2 expression plasmids (as indicated). (a) Ago1 or Ago2 (anti-Myc) or Dcp1a (anti-GFP) immunoprecipitates were western blotted with anti-Myc or anti-GFP antibodies as indicated. (b) Ago1 or Ago2 (anti-Myc) or Dcp2 (anti-Flag) immunoprecipitates were western blotted with anti-Myc or anti-Flag antibodies as indicated. (c) Extracts were treated with RNaseA before immunoprecipitation of Ago2 (anti-Myc) or Dcp1a (anti-GFP). Immunocomplexes were western blotted with anti-Myc or anti-GFP antibodies as indicated. (d) Extracts were treated with RNaseA before immunoprecipitation of Ago2 (anti-Myc) or Dcp2 (anti-Flag). Immunocomplexes were western blotted with anti-Myc or anti-Flag antibodies as indicated.

Figure 3

Accumulation of Argonaute proteins in P-bodies requires an intact siRNA-binding domain. (a) Wild-type and mutant Ago2 proteins, Ago2-PAZ9 and Ago2-PAZ10 (as indicated), were expressed as Myc–epitope fusions in cells transfected with a luciferase siRNA. Western blotting with an anti-Myc antibody was used to measure protein expression (upper panel). Small RNA binding was measured by northern blotting of RNA extracted from immunocomplexes (middle panel). Nuclease activity was also measured against a complementary substrate (bottom panel). (b) Subcellular localization of wild-type and non-small RNA binding mutant Ago2 proteins was determined by immunostaining with anti-Myc antibodies. (c) Myc-tagged wild-type or mutant Ago2 proteins were co-expressed with GFP-tagged Dcp1a. Ago2 (anti-Myc) or Dcp1a (anti-GFP) immunoprecipitates were analysed by western blotting with anti-Myc or anti-GFP antibodies, as indicated.

Figure 4

miRNA-dependent localization of target mRNAs to mammalian P-bodies. (a) Plasmids expressing the let-7 target, Myc–Ago2 protein and MS2–YFP–NLS were cotransfected into U2-OS cells. Reporter mRNA was visualized indirectly using the fusion protein. Ago2 protein was visualized using Rhodamine-Red-conjugated anti-Myc. (b) Analyses were identical to in a except that the target mRNA did not contain the lin-41 3′ UTR fragment. (c) The CXCR4 target was co-expressed with MS2–YFP–NLS and Myc–Ago2. Detection was as in a. (d) The CXCR4 reporter was co-expressed with MS2–YFP–NLS and with (upper) or without (lower) Myc–Ago2 in cells that were also transfected with the CXCR4 siRNA. Endogenous Ago2 was visualized with a rabbit anti-Ago2 antibody. Diagrammatic representation targets are shown next to each panel.