Unraveling molecular effects of ADAR1 overexpression in HEK293T cells by label-free quantitative proteomics

Abstract

ADAR1 is a double-stranded RNA (dsRNA) editing enzyme that specifically converts adenosine to inosine. ADAR1 is ubiquitously expressed in eukaryotes and participate in various cellular processes such as differentiation, proliferation and immune responses. We report here a new proteomics study of HEK293T cells with and without ADAR1 overexpression. The up-and down-regulated proteins by ADAR1 overexpression are identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) followed by label-free protein quantification. Totally 1,495 proteins (FDR < 0.01) are identified, among which 211 are up-and 159 are down-regulated for at least 1.5-fold (n D 3, p < 0.05). Gene ontology analysis reveals that these ADAR1-regulated proteins are involved in protein translation and cell cycle regulation. Bioinformatics analysis identifies a closely related network consistent for the protein translation machinery and a tightly connected network through proliferating cell nuclear antigen (PCNA)-interactions. Up-regulation of the proteins in the PCNA-mediated cell proliferation network is confirmed by Western blotting. In addition, ADAR1 overexpression is confirmed to increase cell proliferation in HEK293T cells and A549 cells. We conclude that ADAR1 overexpression modulates the protein translation and cell cycle networks through PCNA-mediated protein-protein interaction to promote cell proliferation in HEK293 cells.

RNA editing-dependent epitranscriptome diversity in cancer stem cells

“…Notably, ADAR1 depletion in hESCs abrogates pluripotency and induces expression of transcripts that govern differentiation 47 . Conversely, enforced ADAR1 expression in a human embryonic kidney cell line (HEK293T) directly regulates the expression of more than 300 proteins involved in protein translation and cell cycle regulation 48 . Together, these studies suggest that the functional impact of changes in the RNA editome may be far-reaching, but this requires further investigation in the context of primary human cell types and tissue-specific stem cells.…”

Functionally selective activation of the dopamine receptor D2 is mirrored by the protein expression profiles

“…In total, the present study identified 5290 PGs in the proteome of HEK293T-D 2S R cells, allowing for comprehensive expression profiling. Guo et al, who tested the influence of overexpression of adenosine deaminase acting on RNA 1 (ADAR1) on the HEK293T proteome, found 1495 proteins in total in non-fractionated whole cell lysates 32 . Besides 1091 proteins that were identified in both studies, 4199 additional proteins were assigned by the present study, which also used non-fractionated whole cell lysates.…”

<p>Identification and functional characterization of Lys-trimethylation of lactate dehydrogenase A</p>

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