WRNIP1 prevents transcription-associated genomic instability

Reviewer #1 (Public Review):

This paper describes the role of WRNIP1 AAA+ ATPase, particularly its UBZ domain for ubiquitin-binding, but not ATPase, to prevent the formation of the R-loop when DNA replication is mildly perturbated. By combining cytological analysis for DNA damage, R-loop, and chromosome aberration with the proximity ligation assay for colocalization of various proteins involved in DNA replication and transcription, the authors provide solid evidence to support the claim. The authors also revealed a distinct role of WRNIP1 in the prevention of R-loop-induced DNA damage from FANCD2, which is inconsistent with the known relationship between WRNIP1 and FANCD2 in the repair of crosslinks.

Reviewer #2 (Public Review):

This paper aims at establishing the role of WRN-interacting protein 1 (WRNIP1) and its UBZ domain (an N-terminal ubiquitin-binding zinc finger domain) on genome instability caused by mild inhibition of DNA synthesis by aphidicolin. The authors used human MRC5 fibroblasts investigated with standard methods in the field. The results clearly showed that WRNIP1 silencing and UBZ-mutation (D37A) increased DNA damage, chromosome aberrations, and transcription-replication conflicts caused by aphidicolin.

The conclusions of the paper are overall well supported by results, however, aspects of some data analyses would need to be clarified and/or extended.

1 The methods (immunofluorescence microscopy and dot-blots) to determine R-loop levels can lack sensitivity and specificity. In particular, since the S9.6 antibody can bind to other structures besides heteroduplex, dot-blot analyses only grossly assess R-loop levels in cellular samples of purified nucleic acids, which are constituted by many different types of DNA/RNA structures.

2 Experimental plan has analyzed the impact of WRNIP1 lack or mutations at steady-state conditions. Thus, the possible role of WRNIP1 at an early step of the mechanism would require some sort of kinetics analysis of the molecular process, therefore not at steady-state conditions. The findings of a co-localization of R-loops and WRNIP1 have been obtained with the S9.6 antibody, which recognizes DNA-RNA heteroduplexes. Since WRNIP1 is known to be recruited at stalled forks and DNA cleavage sites, it is not surprising that WRNIP1 is very close to heteroduplexes, abundant structures at replication forks and cleavage sites. Similar interpretations may also be valid for Rad51/S9.6 co-localization findings.

3 Determination of DNA damage, chromosome aberration, and co-localization data are reported as means of measurements with appropriate statistics. However, the fold-change values relative to corresponding untreated samples are not reported. In some instances, it seems that WRNIP1 silencing or mutations actually reduce or do not affect aphidicolin effects. That leaves open the interpretation of specific results.

Reviewer #3 (Public Review):

Summary:
In the manuscript by Valenzisi et al., the authors report on the role of WRNIP1 to prevent R-loop and TRC-associated DNA damage. The authors claim WRNIP1 localizes to TRCs in response to replication stress and prevents R-loop accumulation, TRC formation, replication fork stalling, and subsequent DNA damage. While the findings are of potential significance to the field, the strength of evidence in support of the conclusions is lacking.

Weaknesses:

1. The authors fail to utilize the proper controls throughout the manuscript in regard to the shWRNIP1, WT, and mutant cell lines. It is unclear why the authors failed to use the shWRNIP1WT line in the comet assay, DNA fiber assay, and the FANCD2 assays. This is a key control for i) the use of only a single shRNA (most studies will use at least 2 different shRNAs) and ii) the use of the mutant WRNIP1 lines. In several figures, the authors only show the effect of the UBZ mutant, but don't include the ATPase mutant or WT for comparison. Including these is essential.

2. The authors use the S9.6 antibody to conclude the loss of WRNIP1 causes more R-loops; however, it has been shown that this antibody detects dsRNA in addition to RNA-DNA hybrids. Accordingly, it cannot be ruled out that the increased S9.6 signal is due to increased dsRNA.

3. Multiple pieces of data do not support the conclusions. For example, Figure 1D shows shWRNIP1 to reduce damage in Aph+DRB cells compared to MRC5SV cells with Aph+DRB. This result suggests that WRNIP1 actually increases DNA damage in stressed cells with transcription blocked. Another result is seen in Figure 4a, where the number of PLA spots (presumably TRCs) increases in the shWRNIP1WT cells with Aph+RNH1 compared to Aph alone. If R-loops are required for TRC accumulation, then the RNH1 should decrease the PLA foci. This result instead suggests that WRNIP leads to increased TRCs in stressed cells with R-loops cleared by RNH1.

4. The data are mostly phenomenological and fail to yield mechanistic insight. For example, the authors state that "it remains unclear whether WRNIP1 is directly involved in the mechanisms of R-loop removal/resolution". Unfortunately, the data presented in this manuscript do not provide new insights into this unresolved question.

5. The authors only show merged images making it impossible to visualize differences in PLA foci.