Co-transcriptional R-loops are long DNA-RNA hybrids that can arise during transcription by RNA Polymerase II. While R-loops serve important physiological functions, such as class switch recombination and promotion of transcription termination, persistent R-loops represent a major source of DNA damage, replication stress and genome instability. Defects in R-loop resolution are seen in neurodegenerative diseases and cancer. Cells have evolved different strategies to prevent the accumulation of pathological R-loops. One such mechanism relies on the conserved THO complex and its associated co-transcriptional factor UAP56 helicase in humans. However, the detailed mechanism how UAP56 functions in R-loop prevention remains to be determined. Our preliminary data have shown that depletion of UAP56 results in R-loop accumulation and R-loop-mediated genome instability. In addition, purified UAP56 is more adept at unwinding RNA-DNA hybrids than RNA-RNA duplex. It also dissociates model R-loops in vitro. Based on these findings, we hypothesize that UAP56 engages and dissociates co-transcriptional R-loop structures to prevent their accumulation in cells. We also hypothesize that the R-loop dissociation activity of UAP56 needs to be strictly regulated during transcription by its binding partner ALYREF and CHTOP, two RNA binding proteins. The overall goal of this R15 proposal is to determine how UAP56 helps eliminate harmful, co-transcriptional R-loops, and how this activity is regulated by its cofactors to maintain genome stability. In this project, we will achieve our goal by three specific aims: (1) examine co- transcriptional R-loop removal and genome maintenance function of UAP56 in cells; (2) determine the R-loop resolution function of UAP56 in vitro; (3) characterize the regulation of UAP56 in R-loop removal by its physiological partner, ALYREF and CHTOP.
In Aim 1, we will examine the R-loop levels and DNA damage in UAP56 depleted cells using a set of cellular and biochemical methods, including immunofluorescence (IF) using the S9.6 monoclonal antibody, DRIP-qPCR in a set of target genes for DNA-RNA hybrid accumulation, bisulfite sequencing of target genes monitoring conversion of cytosines, DRIPc-seq to map R-loops on the genomic scale, comet assay and ?H2AX foci formation.
In Aim 2, with highly purified UAP56 and its helicase dead mutants, we will investigate its association with a series of RNA/DNA and other nucleic acid substrates, as well as its ability to dissociate DNA-RNA hybrids and R-loops, including plasmid-based physiologically relevant R-loops.
In Aim 3, we will purify ALYREF and CHTOP, and define their influence on UAP56?s activity in the unwinding of DNA- RNA hybrids and R-loop resolution in reconstituted biochemical systems and in cells. The results from our project will shed light on the general mechanism of co-transcriptional R-loop processing and genome preservation. We expect our endeavors to contribute toward the development of novel strategies to prevent pathogenic R-loops formation and to treat neurodegenerative disease and cancer caused by their accumulation. ! 1!
The accumulation of non-scheduled, co-transcriptional R-loops are major cause of replication stress, which leads to DNA double strand breaks (DSBs) and genome instability, and are associated with neurodegenerative diseases and cancer. Our preliminary data have provided evidence that UAP56, a co-transcriptional RNA processing helicase, has the ability to unwind DNA-RNA hybrids and R-loops, preventing their accumulation during transcription in cells. The proposed work will support the mission of NIGMS by elucidating the mechanism of co-transcriptional R-loop removal and genome preservation catalyzed by UAP56. ! 1!
