The Npl3 hnRNP prevents R-loop-mediated transcription–replication conflicts and genome instability

Abstract

Transcription is a major obstacle for replication fork (RF) progression and a cause of genome instability. Part of this instability is mediated by cotranscriptional R loops, which are believed to increase by suboptimal assembly of the nascent messenger ribonucleoprotein particle (mRNP). However, no clear evidence exists that heterogeneous nuclear RNPs (hnRNPs), the basic mRNP components, prevent R-loop stabilization. Here we show that yeast Npl3, the most abundant RNA-binding hnRNP, prevents R-loop-mediated genome instability. npl3D cells show transcription-dependent and R-loop-dependent hyperrecombination and genome-wide replication obstacles as determined by accumulation of the Rrm3 helicase. Such obstacles preferentially occur at long and highly expressed genes, to which Npl3 is preferentially bound in wild-type cells, and are reduced by RNase H1 overexpression. The resulting replication stress confers hypersensitivity to double-strand break-inducing agents. Therefore, our work demonstrates that mRNP factors are critical for genome integrity and opens the option of using them as therapeutic targets in anti-cancer treatment.

Transcription and Recombination: When RNA Meets DNA

“…Thus, TAR in yeast THO complex mutants is only observed under S-phase transcription, and replication forks pause or stall in the DNA region in which a hot spot of RNA-DNA hybrid formation at the end of a lacZ gene has been identified (Huertas et al 2006;Wellinger et al 2006). Indeed, the ability of R-loops to negatively affect progression of replication forks has been confirmed in yeast, C. elegans, and bacteria (Gan et al 2011;Castellano-Pozo et al 2012;Santos-Pereira et al 2013). Therefore, it seems that regardless of the type of TAR detected, whether or not stimulated in mRNP biogenesis and export mutants or mediated by R-loops, its origin is linked to a defective replication progression.…”

“…The relevance of R-loops as an intermediate responsible for different forms of instability is, nowadays, supported by an increasing number of reports. These include the demonstration of the formation of R-loops in DT40 chicken and HeLa cells depleted of the splicing factor ASF/SF2 (Li and Manley 2005) or the transcription-dependent instability manifested as gross-chromosomal rearrangements, g-H2AX foci, or hyper-recombination in yeast, Caenorhabditis elegans, and human cell lines mutated in or depleted of a number of factors involved in RNA metabolism (Gomez-Gonzalez and Aguilera 2007; Gonzalez- Aguilera et al 2008;Paulsen et al 2009;El Hage et al 2010;Dominguez-Sanchez et al 2011;Wahba et al 2011;Castellano-Pozo et al 2012;Stirling et al 2012;Gavalda et al 2013;Santos-Pereira et al 2013). Interestingly, different reports have provided evidence that genome instability associated with DNA repeats with the potential to form non-B DNA structures, such as trinucleotide repeats (Grabczyk et al 2007;Lin et al 2010), or common fragile sites (CFSs) in mammalian cells (Helmrich et al 2011) are also dependent on transcription and associates with the formation of R-loops.…”

A double-edged sword: R loops as threats to genome integrity and powerful regulators of gene expression

“…So far, five different mechanisms are thought to regulate R-loop formation (see Fig. 2): (1) RNase H enzyme, which specifically degrades the RNA in RNA/DNA hybrid (for review, see Cerritelli and Crouch 2009); (2) RNA/DNA helicases such as the yeast Sen1 or homologous human Senataxin (Mischo et al 2011;Skourti-Stathaki et al 2011) and the human DHX9 helicase, which also acts on G4 structures (Chakraborty and Grosse 2011); (3) topoisomerases, which relax DNAnegative supercoiling that otherwise causes persistent Rloop formation (Drolet et al 1994(Drolet et al , 1995Tuduri et al 2009;El Hage et al 2010;Yang et al 2014); (4) proteins that prevent R-loop formation, such as mRNA biogenesis (Huertas and Aguilera 2003;Dominguez-Sanchez et al 2011;Castellano-Pozo et al 2012) and processing proteins (Li and Manley 2005;Paulsen et al 2009;Wahba et al 2011;Stirling et al 2012;Santos-Pereira et al 2013); and (5) suppressors of proteins that promote R-loop formation (i.e., Rad51 and AtNDX) (Sun et al 2013;Wahba et al 2013).…”

Targeting Mcl-1 enhances DNA replication stress sensitivity to cancer therapy

“…DSBs can be induced by ionizing radiation, chemotherapeutic drugs, oxidative stress, and replication fork collapse (2). Inappropriate repair of DSBs may result in propagation of deleterious mutations, genomic instability, immune deficiency, cancer predisposition, and accelerated aging or cell death (3)(4)(5).…”