Overall:All in the regulation of ribosome biogenesis. The authors

Overall:All 3 reviewers were pretty much on the same page regarding what experiments needed to be done in order unequivocally conclude a role for FANCI in the regulation of ribosome biogenesis. The authors provided very few of the requested experiments and basically disagreed with most of the reviewers comments. Instead, they attempt to bully their way out of providing the data with a padded rebuttal response containing unscientific arguments such as citing another NAR paper that did not contain the kind of experiments the reviewers asked for. In my opinion this is not up to par with the expectations in the field of ribosome biogenesis nor up to NAR standards of scientific rigor.  Detailed comments:Although the authors wrote a lengthy point by point response to the reviewers comments, they fail to provide the requested key experiments needed to unequivocally demonstrate that FANCI has a direct role in regulating rRNA transcription and most importantly, processing. The evidences presented to support the author’s conclusions are not within the standards for publication in NAR. In agreement with the other reviewers, it is still not clear to what extent the effects observed following FANCI depletion are direct or indirect consequences of inducing a DNA damage response. It has been clearly demonstrated that depletion of FANCI alone causes an increase in basal level of genomic instability and several studies have shown that activation of the DNA damage response leads to downregulation of rDNA transcription.Main concerns remaining:-The authors claim that they have ruled out the possibility that DNA damage is responsible for the phenotypes observed upon depletion of FANCI by including depletion of FANCD2, which also results in spontaneous DNA damage. The problem here is that depletion of FANCI by RNAi also depletes FANCD2 (Fig S3). However, RNAi of FANCD2 does not affect levels of FANCI. Thus, using the FANCI siRNA, the authors are looking at the potential additive effects of knocking down FANCI + FANCD2, which could increase the level of DNA damage and consequently have a greater effect on inhibiting rRNA synthesis. Hence, this is why it is essential to provide supporting evidence that FANCI has a direct role in regulating ribosome biogenesis (metabolic labeling to show delay in rRNA processing, co-sedimentation with pre60S and impaired 60S synthesis by sucrose fractionation).-FANCI binds to DNA in general and plays an essential role in DNA repair, which includes all DNA (nuclear and nucleolar). As the nucleolus is basically formed by rDNA repeats, FANCI could fractionate in the nucleolar fraction due to its role in DNA repair in general and not necessarily have a direct role in regulating rRNA synthesis. The new IF experiments provided on Fig 1 F-O shows that FANCI localizes everywhere in the nucleus and that it is not enriched in the nucleolus. The supplementary deconvolution “movies” are of poor quality and FANCI does not appear to be in the same Z-plane are the nucleolar markers. The co-localization should be done with Pes1 since they interact together they should be in the same nucleolar compartment. This does not appear to be the case from the data presented.-The Northern blot in Fig 3B shows that all rRNA intermediates (PTP, 41S, 32S and 12S) are decreased in FANCI RNAi treated cells compared to control. This could just be the result of decreased rRNA transcription due to DNA damage. If FANCI is involved in a processing step required to generate the 12S rRNA, there should be a delay or accumulation of an earlier rRNA intermediate. The authors do not provide this key piece of data. It is not the norm in the field of ribosome synthesis to show only one blot with one probe to demonstrate a role in rRNA processing. The effects on rRNA observed with FANCI KD are clearly different from the one observed for Pes1 KD. RAMP analysis with one blot or probe is insufficient. Based on Fig 3I, the authors interpreted that FANCD2 KD is involved in 32S processing, suggesting a role in ribosome biogenesis. However, the authors do not really address this coherently. Ex: “Depletion of FANCD2 did not result in a similar processing defect when compared to FANCI depletion…” and in the Discussion : “…depletion of FANCD2 did result in a significant increase in the ratio of the 32S/41S pre-rRNAs (Fig 3I), suggesting that FANCD2 also has a role in ribosome biogenesis. However, while we do find the FANCD2 protein in the nucleolus, unlike FANCI, FANCD2 is not enriched there (Fig. 1 C, D). At present, we cannot distinguish a specialized role for FANCD2 in the nucleolus beyond its role in repairing the DNA that is found there.” So why would FANCI have a role in ribosome biogenesis but not FANCD2. The claim that FANCI is enriched in the nucleolus is overstated. The IF data does not corroborate the enrichment of FANCI in the nucleolus shown by biochemical fractionation.Thus, Northern blots with using several different probes, metabolic labeling, co-purification with pre60S particle are required to conclude a role for FANCI in rRNA processing. Citing the fact that the authors were not asked to perform these experiments for their previous Nature paper is irrelevant. Contrary to their Nature paper, this manuscript is not a large-scale discovery paper attempting to assign a role for 20+ proteins in rRNA synthesis. In this manuscript, there is only one protein studied, known for a role in DNA repair, so it should not be that demanding to provide these experiments.-If FANCI has an important role in 60S biogenesis, its depletion should induce a nucleolar stress with stabilization of p53 and a cell cycle arrest. Particularly if it has such a dramatic affect on translation (Fig 4). The authors claim that “This is not surprising as not all ribosome biogenesis factors or ribosomal proteins activate the nucleolar stress pathway through p53 (2,50,53).” This is not true and the authors are incorrect with the reference cited to make that claim. In Reference 2 (Jayaraman et al NAR 2017), they used HeLa cells to look at p53 stabilization upon NuMA KD. HeLa cells should never by used to assess nucleolar stress and p53 stabilization as they are a HPV positive and express E6 oncoprotein, which inactivates p53. So it is not surprising that they did not observed p53 stabilization. Reference 50 (Tafforeau et al., Mol Cell 2013) has no data at all supporting that claim. Reference 53 looks at ribosomal proteins, which is very different since a lot of them are required to stabilize p53 through their ability to bind to MDM2. In addition, they used an arbitrary cutoff threshold of five fold to mark a significant p53 accumulation, which is considered high. An accumulation of 2-3 fold is sufficient to generate a dramatic transcriptional induction of p21 and cell cycle arrest.-I also agree with one of the reviewer that the second part of the manuscript on the ubiquitination state of the FANCI protein is unrelated to the first part and distracts from addressing the potential role of the FANCI protein in ribosome biogenesis.- Western blots showing KD of USP1 and USP36 are still missing. The author’s response (response 33) is unacceptable and there are plenty of UPS1 and USP36 antibodies commercially available. At least provide RT-qPCR to show the KD. Demonstrating proper knockdown after RNAi is not a superfluous control.

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