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RNA. 2016 Jul;22(7):1085-98. doi: 10.1261/rna.056499.116. Epub 2016 May 19.

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

Author information

1
Department of Pharmacology, Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
2
Department of Biochemistry and Molecular Biology, Southern Illinois University, Carbondale, Illinois 62901, USA.
3
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.

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.

KEYWORDS:

Argonaute2; RNAi; mass spectrometry; nucleus; proteomics

PMID:
27198507
PMCID:
PMC4911916
DOI:
10.1261/rna.056499.116
[Indexed for MEDLINE]
Free PMC Article

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