Characterization of R-Loop–Interacting Proteins in Embryonic Stem Cells Reveals Roles in rRNA Processing and Gene Expression

生物 染色质 细胞生物学 RNA解旋酶A 核糖核酸 RNA结合蛋白 基因 解旋酶 基因表达 遗传学 基因表达调控
作者
Tong Wu,Jennifer Nance,Feixia Chu,Thomas G. Fazzio
出处
期刊:Molecular & Cellular Proteomics [Elsevier]
卷期号:20: 100142-100142 被引量:16
标识
DOI:10.1016/j.mcpro.2021.100142
摘要

Chromatin-associated RNAs have diverse roles in the nucleus. However, their mechanisms of action are poorly understood, in part because of the inability to identify proteins that specifically associate with chromatin-bound RNAs. Here, we address this problem for a subset of chromatin-associated RNAs that form R-loops—RNA–DNA hybrid structures that include a displaced strand of ssDNA. R-loops generally form cotranscriptionally and have important roles in regulation of gene expression, immunoglobulin class switching, and other processes. However, unresolved R-loops can lead to DNA damage and chromosome instability. To identify factors that may bind and regulate R-loop accumulation or mediate R-loop–dependent functions, we used a comparative immunoprecipitation/MS approach, with and without RNA–protein crosslinking, to identify a stringent set of R-loop–binding proteins in mouse embryonic stem cells. We identified 364 R-loop–interacting proteins, which were highly enriched for proteins with predicted RNA-binding functions. We characterized several R-loop–interacting proteins of the DEAD-box family of RNA helicases and found that these proteins localize to the nucleolus and, to a lesser degree, the nucleus. Consistent with their localization patterns, we found that these helicases are required for rRNA processing and regulation of gene expression. Surprisingly, depletion of these helicases resulted in misregulation of highly overlapping sets of protein-coding genes, including many genes that function in differentiation and development. We conclude that R-loop–interacting DEAD-box helicases have nonredundant roles that are critical for maintaining the normal embryonic stem cell transcriptome.

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