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

• Lukas Stalder ^[2] ; Wolf Heusermann ^[2] ; Lena Sokol ^[2] ; Dominic Trojer ^[2] ; Joel Wirz ^[2] ; Justin Hean ^[2] ; Anja Fritzsche ^[2] ; Florian Aeschimann ^[1] ; Vera Pfanzagl ^[2] ; Pascal Basselet ^[2] ; Jan Weiler ^[2] ; Martin Hintersteiner ^[2] ; David V Morrissey ^[3] ; Nicole C Meisner-Kober ^[2]
  1. [1] Friedrich Miescher Institute

     Friedrich Miescher Institute

     Basilea, Suiza

     
  2. [2] Novartis Institutes for Biomedical Research, NIBR Biologics Center, RNAi Therapeutics, Basel, Switzerland
  3. [3] Novartis Institutes for Biomedical Research, NIBR Biologics Center, RNAi Therapeutics, Cambridge, MA, USA

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• Localización: EMBO journal: European Molecular Biology Organization, ISSN 0261-4189, Vol. 32, Nº. 8, 2013, págs. 1115-1127
• Idioma: inglés
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  • 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.