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.

MicroRNA in Control of Gene Expression: An Overview of Nuclear Functions

“…This assumption is in accordance with previous analysis suggesting that the nuclear RISC seems to be smaller than its cytoplasmic counterpart [37]. More recently, using a semi quantitative mass spectrometry approach, Kalantari et al [41] have confirmed the small size of the nuclear RISC, showing that the association of AGO2 with Gw182/TNRC6 (-A, -B, -C) and AGO3 is best preserved and more stable in both nuclei and cytoplasm whereas the AGO2 interaction with Dicer and TRBP is confined to the cytoplasm. Obviously, different conditions for biochemical purification may yield diverse compositions of the RISC.…”

“…In fact, silencing of IPO8 reduces the pool of nuclear AGO2 [38]. AGO2 is lacking a nuclear localization signal unlike many components of the cytoplasmic RISC, including TNRC6 paralogs and Dicer [39,40,41,42,43], although a recent paper questions Dicer nuclear compartmentalization [44]. However, AGO2 overcomes this deficiency by combining with TNRC6A which possesses a nuclear localization signal (NLS) rather than a nuclear export signal (NES), and works as a nuclear–cytoplasmic shuttling protein to re-localize minimal miRISC to the nucleus [39,45].…”

Synthesis and Evaluation of Parenchymal Retention and Efficacy of a Metabolically Stable O-Phosphocholine-N-docosahexaenoyl-l-serine siRNA Conjugate in Mouse Brain

“…We also observed a small fraction of nuclear-localized PC-DHA-hsiRNAs. Active nuclear RISC complexes have been reported previously 45–46 . The slow kinetics of delivery were analogous to the first generation of DHA-hsiRNA conjugates 47 .…”

Human GW182 Paralogs Are the Central Organizers for RNA-Mediated Control of Transcription

“…Because of its established importance as an RNAi factor, we previously examined the potential for AGO2 to interact with proteins in cell nuclei. Mass spectrometry of the nuclear partners of AGO2, however, revealed only limited insights (Kalantari et al, 2016b). The GW182 paralogs TNRC6A, TNRC6B, and TNRC6C and the AGO variant AGO3 were the only partner proteins detected.…”