MRE11-RAD50-NBS1 activates Fanconi Anemia R-loop suppression at transcription-replication conflicts

Background

R-loops, transcription byproducts consisting of an RNA:DNA hybrid and the resulting single stranded DNA, have both gene regulatory function as well as pose a threat to genome stability. Cells have evolved several mechanisms to regulate R-loop formation, such as occlusion of newly transcribed RNA by RNA processing factors, degradation of RNA:DNA hybrids by RNaseH, or regulation of DNA accessibility by topoisomerase. In addition to these factors, it is becoming apparent that factors in the replication fork are also key R-loop regulators. This is especially important as R-loops are an endogenous source of transcription-replication conflicts. However, a complete understanding of all factors involved and their mechanistic roles is still lacking.

Key Findings

A key component of the double strand break repair pathway, MRE11-RAD50-NBS1 (MRN complex), is also required to process R-loops via recruitment and activation of the Fanconi Anemia pathway.

The authors begin by conducting a screen in yeast to determine factors that impair yeast growth when deleted in combination with both of the catalytic RNaseH enzymes (rnh1∆rnh201∆geneX∆). Among the ~100 hits, the authors find several previously characterized R-loop related proteins. They also find novel factors – including several components of the MRN complex. To characterize the role of MRN complex in R-loop formation, the authors rely on the S9.6 antibody that specifically recognizes RNA:DNA hybrids with subnanomolar affinity. By microscopy and ChIP-qPCR, they demonstrate that knock-down of RAD50 results in increased R-loop accumulation in yeast and mammalian cells. As a control, they validate that overexpression of RnaseH1 abolishes the increase in R-loop accumulation. Next, the authors show that in mammalian cells RAD50 knock-down induces replication stress in an RNH1-dependent manner; RAD50 knock-down activates the ATR DNA response pathway (measured by increase in Chk1 phosphorylation), increases transcription-replication conflicts (measured by decreased proximity between RNAPII-PCNA), and slows replication progression (measured by reduced tract length of IdU/CldU DNA comb labeling).

Additionally, the authors find that the new role of the MRN complex is not dependent on the nuclease activity of Mre11, as treatment with a Mre11 inhibitor or a catalytically inactive Mre11 mutant does not increase R-loop accumulation. Rather, the MRN complex works as a structural element to recruit other factors capable of resolving R-loops. They describe a role for MRN complex in the Fanconia Anemia pathway through epistasis analysis. They further confirm previous reports that MRN recruits the helicases FANCM and BLM. Lastly, they demonstrate that MRN promotes the ubiquination of PCNA by RAD18. RAD18 was a clear choice for further study as it was also a hit in their yeast screen and is an early step of the FA pathway.

Figure 1. Model of MRN function in R-loop tolerance and mitigation (Figure 7 of pre-print).

Importance

R-loops and their role in genome stability are beginning to be understood. This is especially important as genome stability is a hallmark of cancer and other human disorders. In this work, the authors show for the first time that the MRN complex, specifically RAD50, prevents R-loop accumulation in yeast and mammalian cells. The authors do a particularly good job of performing R-loop characterization and replication stress experiments with the additional control of RNH1 overexpression to confirm that any observed effects are R-loop dependent. They then demonstrate that the MRN complex prevents R-loop accumulation through its role in the FA pathway. While a link between MRN and the FA pathway was already known, the authors expand on our mechanistic understanding of how MRN regulates FA factor recruitment by modulating PCNA-ub through RAD18. It is encouraging to see a preprint not only identify a novel factor in R-loop biology, but to begin to link it into the larger context of what is known about R-loop regulation and human disease.

Future directions and questions for the authors

• Do RAD50 and NBS1 have the same effect? Interestingly, only rad50 and xrs2, but not mre11 were identified in the authors original yeast screen, suggesting that MRE11 is not required for R-loop regulation. The authors further demonstrate that knock-down of MRE11 in a RNaseH2 deficient cell line does not impair viability, nor does a catalytically inactive mutant increase R-loop formation. However, the authors do not investigate if there are different requirements of the other components of the MRN complex RAD50 and NBS1. It will be interesting to see future work that starts to disentangle this complex further.
• Is the decreased recruitment of helicases a direct effect of MRN depletion or a downstream consequence of decreased PCNA-ub? Interestingly, the authors show that MRN depletion affects an early step in the FA pathway. Is depletion of RAD18 epistatic to depletion of FANCD2 when combined with RAD50 depletion?
• How does MRN promote PCNA-ub if RAD18 is already primed at R-loop forming loci? The authors note that RAD18 recruitment is not affected by depletion of the MRN complex, just its ubiquitination activity. Further, rad18∆ was an independent hit from their yeast screen. It would be interesting to delve deeper into the role of RAD18 for future studies.

Tags: dsb repair, mre11, r-loop, rad50, yeast

Posted on: 21 January 2019

doi: https://doi.org/10.1242/prelights.7681

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