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

Abstract

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Integrative analysis and prediction of human R-loop binding proteins

“…To develop a consensus dataset, we first intersected 3 studies that document R-loop interacting proteins using orthogonal enrichment approaches followed by mass spectrometry (IP-MS and Prox-MS). Upon intersecting the Cristini et al (2018) dataset (469 proteins), Wang et al (2018) dataset (313; subset of proteins attracted to R-loops), and Wu et al (2021) dataset (364 proteins) we found 49 high confidence proteins that preferentially bind R-loops. Additionally, we also chose common proteins between any 2 studies to reveal a highly stringent list of 292 proteins that we classify as RLBPs identified through IP-MS approaches (IP-MS RLBPs) ( Fig.…”

“…Datasets for curating the IP-MS RLBPs list were taken from Cristini et al (2018) , the R-loop attracted proteins dataset from Wang et al (2018) and Wu et al (2021 ) while the Prox-MS RLBPs list were taken from Mosler et al (2021) and Yan et al (2022 ). Protein domain information was extracted from Pfam version 32.0 using an E value <0.001 ( El-Gebali et al 2019 ).…”

“…This study in HeLa cells identified hundreds of candidate R-loop interacting proteins including many known interactors, and focused on validation of DHX9 as a novel R-loop reader in transcription termination, and eraser in genome stability maintenance ( Cristini et al 2018 ). A similar approach was followed in mouse embryonic stem cells to reveal nonredundant roles of DEAD-box proteins in R-loop modulation ( Wu et al 2021 ), and a related study used synthetic RNA:DNA hybrids conjugated to beads to isolate hybrid binding proteins from B-cell lysates ( Wang et al 2018 ). Overall, these 3 studies that rely on immunoprecipitation coupled with mass spectrometry (IP-MS) identified hundreds of candidate’s RLBPs and overlapped by >200 proteins creating a higher confidence set of proteins that may preferentially recognize R-loops.…”

Role of Nuclear Non-Canonical Nucleic Acid Structures in Organismal Development and Adaptation to Stress Conditions

“…These studies, together with the fact that R-loops are detected at repetitive Satellite sequences in mammalian cells ( Zeng et al, 2021 ), suggest that R-loop dynamics during mammalian development might support gene expression changes during this process. Mouse embryonic stem cells (mESC) have been used to identify R-loop interactors using immunoprecipitation with the S9.6 antibody followed by mass spectrometry ( Wu et al, 2021 ). RBP1, a subunit of RNA polymerase II, and DHX9, RNA helicase, were among the already known R-loop interactors and regulators identified in this study.…”

“…Some proteins of the DEAD-box family, which are known to be involved in R-loop regulation and bind RNAs were also enriched ( Cargill et al, 2021 ). Interestingly, the knockdown of several members of the DEAD-box family leads to the overexpression of genes involved in cell fate commitment and neuronal differentiation ( Wu et al, 2021 ), suggesting that the R-loops regulated by these proteins may be important for the pluripotency of mESC.…”