Characterization of R-loop-interacting proteins in embryonic stem cells reveals roles in ribosomal RNA processing and gene expression

ABSTRACT: Chromatin-associated RNAs have diverse roles in the nucleus. However, their mechanisms of action are poorly understood, in part due to 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 single-stranded DNA. R-loops generally form co-transcriptionally 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 potentially regulate R-loop accumulation or mediate R-loop-dependent functions, we used a comparative immunoprecipitation/mass spectrometry approach, with and without RNA-protein crosslinking, to identify a stringent set of R-loop-binding proteins in mouse embryonic stem cells. We identified 365 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 ribosomal RNA 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 non-redundant roles that are critical for maintaining the normal embryonic stem cell transcriptome.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Embryonic Stem Cell

SUBMITTER: Feixia Chu  

LAB HEAD: Thomas G. Fazzio, Feixia Chu

PROVIDER: PXD022697 | Pride | 2021-09-17

REPOSITORIES: Pride