title: Stable association of RNAi machinery is conserved between the cytoplasm and nucleus of human cells.
abstract: Argonaute 2 (AGO2), the catalytic engine of RNAi, is typically associated with inhibition of translation in the cytoplasm. AGO2 has also been implicated in nuclear processes including transcription and splicing. There has been little insight into AGO2's nuclear interactions or how they might differ relative to cytoplasm. Here we investigate the interactions of cytoplasmic and nuclear AGO2 using semi-quantitative mass spectrometry. Mass spectrometry often reveals long lists of candidate proteins, complicating efforts to rigorously discriminate true interacting partners from artifacts. We prioritized candidates using orthogonal analytical strategies that compare replicate mass spectra of proteins associated with Flag-tagged and endogenous AGO2. Interactions with TRNC6A, TRNC6B, TNRC6C, and AGO3 are conserved between nuclei and cytoplasm. TAR binding protein interacted stably with cytoplasmic AGO2 but not nuclear AGO2, consistent with strand loading in the cytoplasm. Our data suggest that interactions between functionally important components of RNAi machinery are conserved between the nucleus and cytoplasm but that accessory proteins differ. Orthogonal analysis of mass spectra is a powerful approach to streamlining identification of protein partners.
authors.affiliation: Department of Pharmacology, Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
authors.forename: Roya
authors.initials: R
authors.lastname: Kalantari
authors.affiliation: Department of Pharmacology, Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
authors.forename: Jessica A
authors.initials: JA
authors.lastname: Hicks
authors.affiliation: Department of Pharmacology, Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
authors.forename: Liande
authors.initials: L
authors.lastname: Li
authors.affiliation: Department of Biochemistry and Molecular Biology, Southern Illinois University, Carbondale, Illinois 62901, USA.
authors.forename: Keith T
authors.initials: KT
authors.lastname: Gagnon
authors.affiliation: Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
authors.forename: Viswanadham
authors.initials: V
authors.lastname: Sridhara
authors.affiliation: Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
authors.forename: Andrew
authors.initials: A
authors.lastname: Lemoff
authors.affiliation: Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
authors.forename: Hamid
authors.initials: H
authors.lastname: Mirzaei
authors.affiliation: Department of Pharmacology, Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
authors.forename: David R
authors.initials: DR
authors.lastname: Corey
mesh_terms: D060565:Argonaute Proteins / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry; D011485:Protein Binding; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism*
chemical_list: C466422:AGO2 protein, human; D060565:Argonaute Proteins; D034741:RNA, Small Interfering
keywords: Argonaute2; RNAi; mass spectrometry; nucleus; proteomics