R-loops: formation, function, and relevance to cell stress

Abstract

Exposure of genomic, single-stranded DNA (ssDNA) during transcription and replication creates opportunities for the formation of inappropriate secondary structures. Cells manage this exposure by using topoisomerases and helicases to reduce the inherent topological stress that arises from unwinding the double helix and by coating ssDNA with protective protein complexes. Interestingly, specific DNA-RNA hybrids, known as R-loops, form during transcription and exist in homeostasis throughout the genomes of prokaryotes and eukaryotes. These hybrids nucleate from guanine rich clusters in the template strand and extend across GC rich spans of transcribed genes. In vivo regulatory functions have evolved from R-loops, including regulation of gene expression and telomere lengthening. However, they also exist as a form of stress, particularly when replication forks collide with the transcription machinery. New methodologies and models are being developed to delineate the biology of R-loops, including those related to cell stress-based diseases like cancer. As accumulation of R-loops is associated with disease, targeting molecular pathways that regulate their formation or removal could provide new avenues for therapeutic intervention. This review covers recent understandings of the molecular basis for R-loop formation, removal, and biological outcomes in the context of cellular stress.

Keywords: DNA damage; R-loops; RNA-DNA hybrid; ataxia; cancer; chromatin modification; gene transcription; genome instability; mRNA splicing; replication fork stalling.

Figure 1. FIGURE 1: Model of R-loop homeostasis.

A homeostasis exists between formation and removal of R-loops across the genome. Normally, mRNP biogenesis machinery targets nascent RNA and processes and prepares the strand for nuclear export (top). Topoisomerases and Helicases aid in preventing R-loop formation by reducing exposure times of ssDNA during transciption. RNase H removes R-loops by specifically digesting RNA in helical formation. Together these processes reduce R-loop accumulation during transcription. Factors that lead to pausing of RNA polymerase render conditions favorable for R-loop formation (replication stress, DNA damage, fork collisions, etc). R-loops nucleate from G-clusters (dashed green line). The loop then extends along GC rich sequences within the gene during elongation (bottom). The balance between removal and formation creates an equilibrium at nucleation sites across the genome.

Figure 2. FIGURE 2: Biological consequences of R-loop formation.

Formation of R-loops can result in defects in transcription and genomic instability. (A) Evidence indicates that R-loop formation contributes to trinucleotide expansion related diseases. The expansion of GC rich sequence in the gene body creates conditions that are favorable for R-loops and can subsequently cause reduced expression of specific genes (FXN, HTN, ATXN1/2). These diseases typically result in neurodegenerative disorders and ataxia. (B) Head-on collisions between the replication fork and transcription bubble create favorable conditions for R-loop formation. The FA complex, along with BRCA1, and other factors promote bypass and resolve R-loops during these collisions in order to complete replication and maintain genome stability. Mutation in FA complex subunits increase DNA damage associated with collisions and this damage is associated with cancer progression.