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 mechanisms1. 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 degradation2-7. 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.RNAi was initially characterized as a post-transcriptional gene silencing mechanism in which the experimental introduction of long double-stranded RNAs (dsRNAs) induces sequencespecific destruction of homologous mRNAs (reviewed in ref. 1). RNAi pathways are initiated when dsRNAs are processed by Dicer into siRNAs of 21-26 nucleotides. siRNAs are incorporated into the effector complex RISC. In RISC, the siRNA is bound by an Argonaute protein, which uses the sequence of the siRNA to select and cleave complementary substrates (reviewed in ref. 8).RISC can also silence gene expression by preventing protein synthesis. Genetic studies of Caenorhabditis elegans that are mutant for Dicer forged the initial link between a previously known class of small regulatory RNAs, the stRNAs, and the RNAi pathway9-13. Subsequent studies showed that stRNAs are archetypes of a large class of regulatory RNAs, known as miRNAs (reviewed in ref. 14). Although miRNA and siRNA pathways can be biochemically compartmentalized, both types of RNAs enter RISC, bind to Argonaute proteins and identify their silencing targets in conceptually similar ways. They differ, at least in animals, in that miRNAs most often pair imperfectly with their targets and are thus unable to direct Argonautemediated cleavage15. Instead, miRNAs repress protein synthesis in a cleavage-independent fashion8,14,15.3 Correspondence should be addressed to G.J.H. (e-mail: hannon@cshl.org) and R.P. (e-mail: rrparker@u.arizona.edu).Note added in proof: After this study had been accepted for publication, Sen and Blau30 reported similar observations of the localization of human Ago2 to cytoplasmic P-bodies.Note: Supplementary Information is available on the Nature Cell Biology website. The mechanism by which miRNAs repress translation of their target mRNAs is unknown. Conceivably, RISC could prevent protein synthesis from miRNA targets in one of several ways. RISC could affect translation, per se, by altering rates of initiation or elongation by the riboso...

Detection of the argonaute protein Ago2 and microRNAs in the RNA induced silencing complex (RISC) using a monoclonal antibody

“…The human Ago proteins have 77.6 to 83.6% sequence identity with each other (Sasaki et al, 2003) and 4F9 most likely recognizes a shared conformational epitope found in all Ago proteins. In addition, it was previously reported that all 4 Ago proteins localized to GWBs (Liu et al, 2005b). Taken together, we concluded that 4F9 binds all Ago proteins.…”

“…The key components of the RISC complex are Argonaute (Ago) proteins with molecular masses of approximately 100 kDa and containing PAZ and PIWI domains (Carmell et al, 2002;Sasaki et al, 2003). Four of these, Ago1 to Ago4, have been demonstrated to associate with miRNAs in humans (Liu et al, 2005b). However, only Ago2 has been demonstrated to possess the activity of miRNA-guided mRNA cleavage or translational inhibition .…”

Principles and effects of microRNA-mediated post-transcriptional gene regulation

“…Moreover, AIN-1, the worm GW182 orthologue, interacts with DCR-1 suggesting that events before RISC assembly may take place in these structures (Ding et al, 2005;Meister et al, 2005). The RNA-and miRNA-independent interaction between hAgo2 and Dcp1a strengthens this hypothesis (Liu et al, 2005b).…”

Molecular medicine of microRNAs: structure, function and implications for diabetes

“…Within P-bodies, miRNA/ mRNA-bound argonaute protein recruits GW182 protein (TNRC6A), which subsequently recruits deadenylating enzyme CCR4-NOT1 (CNOT1), and this is followed by mRNA decapping by DCP1-DCP2 enzyme -thereby affecting stability of repressed mRNA. Repressed mRNAs are then degraded by 5 0 to 3 0 exonuclease activity of XRN1 (5 0 -exoribonuclease 1) (Refs 43,55,57,62,63) (Fig. 2c).…”