Despite their widespread roles as regulators of gene expression, important questions remain about target regulation by microRNAs. Animal microRNAs were originally thought to repress target translation, with little or no influence on mRNA abundance, whereas the reverse was thought to be true in plants. Now, however, it is clear that microRNAs can induce mRNA degradation in animals and, conversely, translational repression in plants. Recent studies have made important advances in elucidating the relative contributions of these two different modes of target regulation by microRNAs. They have also shed light on the specific mechanisms of target silencing, which, although it differs fundamentally between plants and animals, shares some common features between the two kingdoms.

MicroRNAs (miRNAs) are a conserved class of small non-coding RNAs that assemble with Argonaute proteins into miRNA-induced silencing complexes (miRISCs) to direct post-transcriptional silencing of complementary mRNA targets. Silencing is accomplished through a combination of translational repression and mRNA destabilization, with the latter contributing to most of the steady-state repression in animal cell cultures. Degradation of the mRNA target is initiated by deadenylation, which is followed by decapping and 5'-to-3' exonucleolytic decay. Recent work has enhanced our understanding of the mechanisms of silencing, making it possible to describe in molecular terms a continuum of direct interactions from miRNA target recognition to mRNA deadenylation, decapping and 5'-to-3' degradation. Furthermore, an intricate interplay between translational repression and mRNA degradation is emerging.

MicroRNAs (miRNAs) silence the expression of target genes post-transcriptionally. Their function is mediated by the Argonaute proteins (AGOs), which colocalize to P-bodies with mRNA degradation enzymes. Mammalian P-bodies are also marked by the GW182 protein, which interacts with the AGOs and is required for miRNA function. We show that depletion of GW182 leads to changes in mRNA expression profiles strikingly similar to those observed in cells depleted of the essential Drosophila miRNA effector AGO1, indicating that GW182 functions in the miRNA pathway. When GW182 is bound to a reporter transcript, it silences its expression, bypassing the requirement for AGO1. Silencing by GW182 is effected by changes in protein expression and mRNA stability. Similarly, miRNAs silence gene expression by repressing protein expression and/or by promoting mRNA decay, and both mechanisms require GW182. mRNA degradation, but not translational repression, by GW182 or miRNAs is inhibited in cells depleted of CAF1, NOT1, or the decapping DCP1:DCP2 complex. We further show that the N-terminal GW repeats of GW182 interact with the PIWI domain of AGO1. Our findings indicate that GW182 links the miRNA pathway to mRNA degradation by interacting with AGO1 and promoting decay of at least a subset of miRNA targets.[Keywords: Argonaute; deadenylation; decapping; GW182; mRNA decay; P-bodies] Supplemental material is available at http://www.genesdev.org. RNA silencing pathways are conserved, and regulate gene expression at both transcriptional and post-transcriptional levels. These pathways are triggered by the presence of double-stranded RNAs (dsRNAs) of diverse origin, which are processed into small RNAs of 20-30 nucleotides in length. MicroRNAs (miRNAs) are a specific class of genome-encoded small RNAs that regulate gene expression post-transcriptionally (Bartel 2004;Filipowicz 2005). We now know of hundreds of miRNAs in different organisms, and each is thought to regulate the expression of hundreds of targets. They are implicated in biological processes as diverse as cell differentiation and proliferation, apoptosis, metabolism, and development (Bartel 2004;Filipowicz 2005).To accomplish their regulatory function miRNAs associate with the Argonaute proteins to form RNA-induced silencing complexes (RISCs), which elicit decay or translational repression of complementary mRNA targets (Bartel 2004;Filipowicz 2005). In plants, miRNAs are often fully complementary to their targets, and elicit mRNA decay. In contrast, animal miRNAs are only partially complementary to their targets, and silence gene expression by mechanisms that remain elusive (Filipowicz 2005). Recent studies have shown that miRNAs silence gene expression by inhibiting translation initiation at an early stage involving the cap structure, as mRNAs translated via cap-independent mechanisms escape miRNA-mediated silencing (Humphreys et al. 2005;Pillai et al. 2005). Other studies have suggested that translation inhibition occurs after initiation, based on the observation that miRNAs an...

Post-transcriptional processes have a central role in the regulation of eukaryotic gene expression. Although it has been known for a long time that these processes are functionally linked, often by the use of common protein factors, it has only recently become apparent that many of these processes are also physically connected. Indeed, proteins that are involved in mRNA degradation, translational repression, mRNA surveillance and RNA-mediated gene silencing, together with their mRNA targets, colocalize within discrete cytoplasmic domains known as P bodies. The available evidence indicates that P bodies are sites where mRNAs that are not being translated accumulate, the information carried by associated proteins and regulatory RNAs is integrated, and their fate - either translation, silencing or decay - is decided.

P bodies are cytoplasmic domains that contain proteins involved in diverse posttranscriptional processes, such as mRNA degradation, nonsense-mediated mRNA decay (NMD), translational repression, and RNAmediated gene silencing. The localization of these proteins and their targets in P bodies raises the question of whether their spatial concentration in discrete cytoplasmic domains is required for posttranscriptional gene regulation. We show that processes such as mRNA decay, NMD, and RNA-mediated gene silencing are functional in cells lacking detectable microscopic P bodies. Although P bodies are not required for silencing, blocking small interfering RNA or microRNA silencing pathways at any step prevents P-body formation, indicating that P bodies arise as a consequence of silencing. Consistently, we show that releasing mRNAs from polysomes is insufficient to trigger P-body assembly: polysome-free mRNAs must enter silencing and/or decapping pathways to nucleate P bodies. Thus, even though P-body components play crucial roles in mRNA silencing and decay, aggregation into P bodies is not required for function but is instead a consequence of their activity.