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The Core RNAi Machinery is Conserved Between the Cytoplasm and Nuclei of Human Cells

ABSTRACT: Argonaute 2 (AGO2), the catalytic engine of RNAi, is typically associated with inhibition of translation in the cytoplasm. Contrary to dogma, 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 the core RNAi machinery is 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.

INSTRUMENT(S): QExactive Plus, Orbitrap Elite, Q Exactive

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: David Corey

PROVIDER: MSV000079508 | MassIVE | Thu Feb 11 12:40:00 GMT 2016

SECONDARY ACCESSION(S): PXD003619

REPOSITORIES: MassIVE

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