The rough endoplasmatic reticulum is the central site of siRNA-mediated RNA silencing

Abstract

Despite the rapid advancement of our mechanistic understanding of the RNA interference (RNAi) pathways in the past years, the subcellular sites of RNA silencing still remain under debate. Here we show that a lion’s share of transfected small interfering RNA (siRNA) is cleared quickly and only few siRNA molecules are finally getting loaded into Ago2, with as little as 20 30 siRISC molecules per cell sufficient to promote 50 % mRNA knockdown. While the major RNAi pathway proteins are found in most subcellular compartments, the microRNA (miRNA)- and siRNA loaded Ago2 population as well as the RNAi mediated mRNA cleavage product co-sediment exclusively with the membranes of the rough endoplasmatic reticulum (rER) together with the RISC loading complex (RLC) factors Dicer, TRBP and PACT. Moreover, siRNA-loaded Ago2 associates with the cytosolic side of membranes through TRBP and PACT in an RNA-independent manner, potentially mediated through indirect interaction via Dicer. Our findings demonstrate that the outer membrane of the rER is the central site of RNA silencing, which explains the remarkable thermodynamic and kinetic efficiency of this mechanism