Despite progress in mechanistic understanding of the RNA interference (RNAi) 
pathways, the subcellular sites of RNA silencing remain under debate. Here we 
show that loading of lipid-transfected siRNAs and endogenous microRNAs (miRNA) 
into RISC (RNA-induced silencing complexes), encounter of the target mRNA, and 
Ago2-mediated mRNA slicing in mammalian cells are nucleated at the rough 
endoplasmic reticulum (rER). Although the major RNAi pathway proteins are found 
in most subcellular compartments, the miRNA- and siRNA-loaded Ago2 populations 
co-sediment almost exclusively with the rER membranes, together with the RISC 
loading complex (RLC) factors Dicer, TAR RNA binding protein (TRBP) and protein 
activator of the interferon-induced protein kinase (PACT). Fractionation and 
membrane co-immune precipitations further confirm that siRNA-loaded Ago2 
physically associates with the cytosolic side of the rER membrane. Additionally, 
RLC-associated double-stranded siRNA, diagnostic of RISC loading, and 
RISC-mediated mRNA cleavage products exclusively co-sediment with rER. Finally, 
we identify TRBP and PACT as key factors anchoring RISC to ER membranes in an 
RNA-independent manner. Together, our findings demonstrate that the outer rER 
membrane is a central nucleation site of siRNA-mediated RNA silencing.