title: The rough endoplasmatic reticulum is a central nucleation site of siRNA-mediated RNA silencing.
abstract: 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.
authors.affiliation: Novartis Institutes for Biomedical Research, NIBR Biologics Center, RNAi Therapeutics, Basel 4000, Switzerland. lukas.stalder@sakk.ch
authors.forename: Lukas
authors.initials: L
authors.lastname: Stalder
authors.forename: Wolf
authors.initials: W
authors.lastname: Heusermann
authors.forename: Lena
authors.initials: L
authors.lastname: Sokol
authors.forename: Dominic
authors.initials: D
authors.lastname: Trojer
authors.forename: Joel
authors.initials: J
authors.lastname: Wirz
authors.forename: Justin
authors.initials: J
authors.lastname: Hean
authors.forename: Anja
authors.initials: A
authors.lastname: Fritzsche
authors.forename: Florian
authors.initials: F
authors.lastname: Aeschimann
authors.forename: Vera
authors.initials: V
authors.lastname: Pfanzagl
authors.forename: Pascal
authors.initials: P
authors.lastname: Basselet
authors.forename: Jan
authors.initials: J
authors.lastname: Weiler
authors.forename: Martin
authors.initials: M
authors.lastname: Hintersteiner
authors.forename: David V
authors.initials: DV
authors.lastname: Morrissey
authors.forename: Nicole C
authors.initials: NC
authors.lastname: Meisner-Kober
mesh_terms: D060565:Argonaute Proteins / Q000032:analysis; D053487:DEAD-box RNA Helicases / Q000032:analysis; D004721:Endoplasmic Reticulum / Q000737:chemistry / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000032:analysis; D043244:Ribonuclease III / Q000032:analysis; D000099253:Trans-Activation Responsive RNA-Binding Protein
chemical_list: D060565:Argonaute Proteins; D053487:DEAD-box RNA Helicases; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D043244:Ribonuclease III; D000099253:Trans-Activation Responsive RNA-Binding Protein; C466422:AGO2 protein, human; C104647:PRKRA protein, human; C408087:DICER1 protein, human