Characterization of R-Loop-Interacting Proteins in Embryonic Stem Cells Reveals Roles in rRNA Processing and Gene Expression.
Mol Cell Proteomics
; 20: 100142, 2021.
Article
em En
| MEDLINE
| ID: mdl-34478875
ABSTRACT
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.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
RNA Helicases DEAD-box
/
Células-Tronco Embrionárias Murinas
/
Estruturas R-Loop
Tipo de estudo:
Prognostic_studies
Limite:
Animals
Idioma:
En
Revista:
Mol Cell Proteomics
Assunto da revista:
BIOLOGIA MOLECULAR
/
BIOQUIMICA
Ano de publicação:
2021
Tipo de documento:
Article
País de afiliação:
Estados Unidos