In animals, P-bodies or GW-bodies appear to cause the congregation of proteins involved in microRNA (miRNA)-mediated post-transcriptional silencing. The localization of P-bodies does not overlap with that of known organelles and are thus considered independent of lipid bilayers. Nonetheless, an miRNA effector protein, argonaute 2 (AGO2), was initially identified as membrane-associated, and some miRNAs have been found in secreted vesicles (exosomes) that derive from endo-lysosomal compartments called multivesicular bodies (MVBs). Proteins can be sorted in a ubiquitin-dependent manner into MVBs by three heteromeric subcomplexes, collectively termed ESCRT (endosomal sorting complex required for transport), to be further secreted in exosomes and/or degraded by the lysosome. Here we show that GW-bodies containing GW182 and AGO2, two main components of the RNA-induced silencing complex (RISC), are distinct from P-bodies due to their congregation with endosomes and MVBs. Moreover, miRNAs and miRNA-repressible mRNAs are enriched at these cellular membranes, suggesting that endosomes and/or MVBs are sites of miRNA-loaded RISC (miRISC) accumulation and, possibly, action. We further show that purified exosome-like vesicles secreted by MVBs are considerably enriched in GW182, but not P-body components, AGO2 or miRNA-repressible mRNA. Moreover, cells depleted of some ESCRT components show compromised miRNA-mediated gene silencing and over-accumulate GW182, which associates with ubiquitylated proteins. Therefore, GW182, possibly in association with a fraction of miRNA-loaded AGO2, is sorted into MVBs for secretion and/or lysosomal degradation. We propose that this process promotes continuous assembly or disassembly of membrane-associated miRISCs, which is possibly required for miRNA loading or target recognition and subsequent silencing.

In antiviral RNA interference (RNAi), the DICER enzyme processes virus-derived double-stranded RNA (dsRNA) into small interfering RNAs (siRNAs) that guide ARGONAUTE proteins to silence complementary viral RNA. As a counterdefense, viruses deploy viral suppressors of RNAi (VSRs). Well-established in plants and invertebrates, the existence of antiviral RNAi remains unknown in mammals. Here, we show that undifferentiated mouse cells infected with encephalomyocarditis virus (EMCV) or Nodamura virus (NoV) accumulate ~22-nucleotide RNAs with all the signature features of siRNAs. These derive from viral dsRNA replication intermediates, incorporate into AGO2, are eliminated in Dicer knockout cells, and decrease in abundance upon cell differentiation. Furthermore, genetically ablating a NoV-encoded VSR that antagonizes DICER during authentic infections reduces NoV accumulation, which is rescued in RNAi-deficient mouse cells. We conclude that antiviral RNAi operates in mammalian cells.

DNA methylation is essential for silencing transposable elements and some genes in higher eukaryotes, which suggests that this modification must be tightly controlled. However, accidental changes in DNA methylation can be transmitted through mitosis (as in cancer) or meiosis, leading to epiallelic variation. We demonstrated the existence of an efficient mechanism that protects against transgenerational loss of DNA methylation in Arabidopsis. Remethylation is specific to the subset of heavily methylated repeats that are targeted by the RNA interference (RNAi) machinery. This process does not spread into flanking regions, is usually progressive over several generations, and faithfully restores wild-type methylation over target sequences in an RNAi-dependent manner. Our findings suggest an important role for RNAi in protecting genomes against long-term epigenetic defects.