Global prediction of candidate R-loop binding and R-loop regulatory proteins

Abstract

In the past decade there has been a growing appreciation for R-loop structures as important regulators of the epigenome, telomere maintenance, DNA repair and replication. Given these numerous functions, dozens, or potentially hundreds, of proteins could serve as direct or indirect regulators of R-loop writing, reading, and erasing. In order to understand common properties shared amongst potential R-loop binding proteins (RLBPs) we mined published proteomic studies and distilled 10 features that were enriched in RLBPs compared to the rest of the proteome. We used these RLBP-specific features along with their amino acid composition to create a random forest classifier which predicts the likelihood of a protein to bind to R-loops. In parallel, we employed a whole-genome CRISPR screen coupled with flow-cytometry using the S9.6 monoclonal antibody to sort guide RNAs associated with induction of high S9.6 staining. Known R-loop regulating pathways such as splicing and DNA damage repair are highly enriched in our datasets, and we validate two new R-loop modulating proteins. Together these resources provide a reference to pursue analyses of novel R-loop regulatory proteins.

Mismatch repair protein MLH1 suppresses replicative stress in BRCA2-deficient breast tumors

“…Next, we used synthetic R-loops and pure MLH1 protein and detected the binding between R-loops and MLH1 by S9.6-mediated ChIP-IB (Figure 4E). Our findings are supported by a recent report that identified MLH1 as an R-loop-binding protein by a global prediction model (32). By in vitro nuclease assay, we observed that MLH1 degraded the RNA strand of R-loop structure with or without tails in a concentration-dependent manner (Figure 4F).…”