Stable association of RNAi machinery is conserved between the cytoplasm and nucleus of human cells

Kalantari Roya, A. Hicks Jessica, L. I. Liande, T. Gagnon Keith, Sridhara Viswanadham, Lemoff Andrew, Mirzaei Hamid, R. Corey David

Research output: Contribution to journalArticle

18 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)1085-1098
Number of pages14
JournalRNA
Volume22
Issue number7
DOIs
StatePublished - Jul 2016

Keywords

  • Argonaute2
  • Mass spectrometry
  • Nucleus
  • Proteomics
  • RNAi

ASJC Scopus subject areas

  • Molecular Biology

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    Roya, K., Jessica, A. H., Liande, L. I., Keith, T. G., Viswanadham, S., Andrew, L., Hamid, M., & David, R. C. (2016). Stable association of RNAi machinery is conserved between the cytoplasm and nucleus of human cells. RNA, 22(7), 1085-1098. https://doi.org/10.1261/rna.056499.116