DDX47, MeCP2, and other functionally heterogeneous factors protect cells from harmful R loops.

Unscheduled R loops can be a source of genome instability, a hallmark of cancer cells. Although targeted proteomic approaches and cellular analysis of specific mutants have uncovered factors potentially involved in R-loop homeostasis, we report a more open screening of factors whose depletion causes R loops based on the ability of activation-induced cytidine deaminase (AID) to target R loops. Immunofluorescence analysis of γH2AX caused by small interfering RNAs (siRNAs) covering 3,205 protein-coding genes identifies 59 potential candidates, from which 13 are analyzed further and show a significant increase of R loops. Such candidates are enriched in factors involved in chromatin, transcription, and RNA biogenesis and other processes. A more focused study shows that the DDX47 helicase is an R-loop resolvase, whereas the MeCP2 methyl-CpG-binding protein uncovers a link between DNA methylation and R loops. Thus, our results suggest that a plethora of gene dysfunctions can alter cell physiology via affecting R-loop homeostasis by different mechanisms.

RNase H1 Hybrid-Binding Domain-Based Tools for Cellular Biology Studies of DNA-RNA Hybrids in Mammalian Cells.

R loops are abundant noncanonical DNA-RNA hybrid structures that can occur during DNA-based processes, such as transcription, replication and DNA damage, and can lead both to physiologically favorable and pathological outcomes. With an increasing body of work feeding the field of R loop biology, our understanding of the processes in which R loops intervene and the consequences of meddling with R loop formation and dissolution has greatly increased but it has also led to new questions and sometimes opposing possibilities. Proper detection of these structures is a crucial factor to advance our knowledge about R loops and factors associated with their formation and removal. Here, we describe the use of fluorescently tagged HBD, the hybrid-binding domain of RNase H1, as a tool for analyzing DNA-RNA hybrids in different contexts using live-cell microscopy and immunofluorescence experiments.