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Referee #1
Evidence, reproducibility and clarity
Summary:
Provide a short summary of the findings and key conclusions (including methodology and model system(s) where appropriate).
Here Ha et al. has further developed their Pumilio RNA tagging methodology for the isolation of UV-crosslinked proteins that are suggested to associate with Xist RNA in mouse embryonic stem cells (mESCs). Within this study the authors claim to have found the Lupus antigen RNA binding protein (La) as a novel Xist interacting partner that influences the efficacy of X-chromosome inactivation (XCI). The authors use a number of different techniques such as qPCR, fluorescent imaging, ATAC-SEQ and SHAPE to show aberration of XCI upon La shRNA knockdown. However, this study has significant flaws in the efficient isolation and validation of Xist associated proteins using their FLAG-out methodology. Furthermore, later experiments predominantly focus on cell death/survival assays, which is somewhat troubling given the essential roles La plays in processes such as cell differentiation and proliferation, ribosome biogenesis, transcriptional control and tRNA maturation. I feel the authors need to robustly address the potential effects La knockdown may be having on their mESCs.
Major comments:
-Are the key conclusions convincing?
My major concern is in their Xist RNA purification. First of all, I couldn't find any data on proving the enrichment of Xist RNA itself in their Pumilio pull-down experiment. It would have been useful to show Xist RNA enrichment before benzonase step. Secondly, it is hard to imagine the protocol would successfully isolated Xist RNA-protein complexes from the cell. An earlier report by Clemson et al., (J Cell Biol., 1996) has shown that majority of Xist RNA is still stuck in the nucleus after nuclear matrix prep protocol using detergent, which is not so different from the authors' protocol. Moreover, the authors used UV crosslink, which would have made even harder to purify Xist RNA without sonication. Thirdly, as the tag is located on 5' of Xist RNA, it is rather surprising to see that Spen is not detected in their pulldown. Spen is one of the main functional interactors with Xist, robustly detected by several previous reports. Similarly, other high-affinity binders of Xist such as hnRNP-K and Ciz1 were also lacking from this screen. Finally, the peptides found associated with FLAG-out Xist are extremely low in comparison with other data using glutaraldehyde or formaldehyde crosslinking. For example, HnRNP-M found in Chu et al 2015 has 1120 peptide counts in differentiated cells. The authors here use HnRNP-M as a baseline for specific interactions and show a total of 6 peptide counts in Xist expressing cells and 5 in i-Empty cells (Supplementary excel sheet 1). Similarly, the La protein of interest in this study has 8 counts in i-FLAG-Xist and 6 counts in i-Empty. I struggle to see how this result indicate specific Xist binding. Worryingly this is the starting rationale for the rest of their experiments, it is hard to therefore accept the rest of their conclusions either.
The other key conclusion the authors make is from the use of numerous cell death/survival assays for both male and female cell lines. This is extremely troubling in the context of assessing their target protein La. La is involved in multiple RNA maturation events of rRNAs, tRNAs and other polIII transcripts. Furthermore, La has been implicated in binding to the mRNA for Cyclin D1 in both human cells and mouse fibroblasts (NIH/3T3 - male) which show a significant effect on cell proliferation upon siRNA knockdown https://www.nature.com/articles/onc2010425. This, along with the observation that La knock-out blastocysts fail to develop any mice or ES cell lines (male or female) show the effect observed in the authors results is most likely not X-linked cell death https://mcb.asm.org/content/mcb/26/4/1445.full.pdf. The authors need to show that their shRNA KD isn't affecting the proliferation and general fitness of their mESC lines.
- Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?
As discussed above, I feel the authors have not clearly demonstrated Xist specific protein enrichment and haven't proven X-linked cell death. Due to the lack of necessary control experiments as discussed below, I feel the notion that La is involved directly in XCI as an RNA chaperone is currently preliminary/speculative.
- Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.
I would suggest them to show RT-qPCR results of Xist RNA enrichment from the sample after flagIP before benzonase treatment.
Also, it would have been more convincing if their negative control construct (i-Empty) would contain 25 copies of PBSb RNA at least.
In Fig1b, the total amount of proteins loaded on the gel is not equivalent between two lanes. The gel should show equivalent amounts of proteins on the gel. It looks like if the negative control sample had been loaded at the same amount as the one with Xist, the band pattern wouldn't be distinguishable between the two samples. Furthermore, as these samples were used in the following mass spectrometry screen it may suggest that the minimal increase in peptide counts observed in the iXist FLAG-out were due to an increased amount of sample being loaded? No controls are conducted to account for this.
The authors quantify cell death in figures 2C - E. It seems clear that shSsb 1 and 2 have an effect on cell count even in the absence of Dox. The rescue effect seen upon Dox addition is minimal when compared to Empty + Dox 2D. The authors ∆A-iXist line with and without Ssb KD/Dox would be an informative control on whether the increase in cell survival that they see is X-linked.
The qPCR results used to validate silencing defects show minor changes in expression and also don't show significant silencing of X-linked genes sufficient for cell death. Could this be because only ~ 50 - 60% of Male iXist cells seem to be expressing in the movies and that this will have an effect on the observed qPCR results? Furthermore, it seems counterintuitive that expression in the Empty male cells increases in 48h compared to 14h. Is this due to cell death and positive selection of cells less able to silence their X-chromosome? How would these data look in the female XX line? How would the data look in a ∆A-iXist line in the presence and absence of shSsb/Dox?
Confusingly, the male line in Fig 3C shows a drop in live cell count at day 6 of differentiation? Surely given their previous results in Fig 2 the Ssb KD should increase cell viability with +Dox? Ssb KD seems to have an adverse effect on ES cells during extended differentiation protocols. In Figure S1 the authors show ~ 8 - 10% survival of male lines during differentiation. Could the recombination of the Xist sequence around the loxP sites enable the cells to outcompete the dead cells? How would iEmpty and ∆A-iXist cells compare here? Have the differentiated cells been tested for their expression of Xist? Additionally, how are there similar live cell counts for male vs female lines when ~90% of male cells die during differentiation? Were more cells plated at day 4? If so, this would bias the competition of male cell survival and therefore make the male line an inappropriate control. Given the essential role of La during development a control is needed to prove that this death is X-linked in the female 3F1 line. For example, an XO cell line retaining the Cast allele and shSsb expression could show the amount of death caused from shSsb alone independent of X-linked cell death.
If I understood correctly, the RNA FISH used dsDNA probes ("Sx9") against 40 kb of the X-inactivation centre (Xic). Surely Tsix or other Xic transcripts will also be visible? Can the authors use their RNA FISH to determine the XX or XO status of their cells? In Figure S5 a number of cells appear to show a single pinpoint of transcription. This could either be low levels of Xist transcripts or Xic transcription from an XO line in which the 129 chromosome is missing. It would be best to solely quantify cells which have two x chromosomes and if a significant amount of X chromosomes have been kicked out, this should be discussed and controlled for.
In Fig6, the authors generated a number of Ssb constructs for a rescue assay. However, these results complicate the matter and raise more questions than they address. It seems odd that the ∆RRM1 does not rescue based on comparison with their putative negative control, ∆NLS. However, the ∆RRM1 + 2 and ∆LAM do rescue the phenotype better than the full length Ssb? This makes no logical sense and highlights the inherent variation in cell viability these generated cell lines seem to show. Following on from this, figure S7 quantifies the GFP tag mRNA levels, depicting all ∆RRM mutants with expression below ~30%? How can ∆RRM1 or 2 be rescuing in this scenario? Have these lines been tested for their XX or XO status? The loss of an X chromosome would lead to a rescue of the cell death phenotype, which is a process known to occur in XX lines that have been cultured for extended periods of time. Could it also be that the cell lines derived are more or less sensitive to exogenous shRNA expression? Also, further validation is needed to assess the efficiency of KD in these lines as theoretically most of these constructs will be targeted by shRNA? What is the endogenous Ssb expression level in these lines? Where in the mRNA sequence are the shRNAs targeted to? Does this make sense on the relative expression levels of ∆RRM1/2 for example? Further testing of GFP expression could also be assessed by quantitative western blot of GFP or even visualised in their RNA FISH/IF samples (Figure S8), currently neither are shown. In addition, some kind of information of stability of each Ssb protein constructs has not been demonstrated.
For the data shown in Figure 7A and B the authors quantify the % of cells with Xist signal. The authors have already shown a defect in Xist visualisation in Ssb KD. Surely it is plausible to assume a faster loss of Xist signal below background in weaker expressing cells. A more appropriate quantification would be the % loss of Xist signal per cell over time.
With Figure 7C and D, the samples have been treated with actinomycin D which globally affects the transcription of cells even the PolIII associated genes Ssb is needed to mature. This treatment could have an added effect on cell mortality and function. Data confirming that actinomycin D doesn't affect the cells disproportionately is needed. The difference in half-life could be attributed to such a treatment.
In summarising the authors claim that La binds Xist to facilitate folding and appropriate spreading of Xist along the X-chromosome. No direct interaction has been shown, CLIP-seq data would resolve this, however I do understand this is a challenging technique. The authors have instead opted for RIP followed by qPCR (Figure S2). However, this process has a greater potential for non-specific recovery of RNAs via indirect binding. Furthermore, qPCR may also amplify the relative abundance of the RNA detected. As multiple nucleolar proteins came down in the mass spec screen and FLAG-Ssb is being over expressed, it is plausible to assume some transient Xist interactions may arise from nucleolar association at which La will be in high abundance. Positive and negative nuclear RNA controls (e.g. 7SK and U1 snRNA respectively) could be used so to determine the amount of non-specific Protein-RNA interactions in their RIP pull downs. Cytoplasmic actin is not an appropriate control as it is cytosolic.
Other than this the authors may want to probe (via IF) for the presence of La accumulation on the X? Many other know factors such as Ciz1, hnrnpK and PRC1/2 complexes show clear accumulation on the X. If I understand correctly, there are many La antibodies on the market and endogenous levels on the X could be assessed. These antibodies may be useful in IP's and pull downs also.
-Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.
The experiments suggested above are centrally focussed on the cell lines that are currently in the authors possession with maybe exceptions with the ∆A-iXist-shSsb line suggested. However, this should be reasonably quick to obtain given their previous work for this paper. Most experiments suggested will focus on the validation of karyotype, Xist expression, rescue construct expression, further RNA FISH classification and repeating more appropriate positive and negative controls for a number of experiments. In theory this can be obtained relatively simply and quickly from current resources. But with the sheer volume of further experiments that are required here, this may take a significant amount of time. One vital improvement needed is the replication of mass spec data and the validation of Xist specific recovery and protein enrichment. As it stands this manuscript seems to not have any replicates of the FLAG-out methodology and mass spec data. This is troubling given the poor recovery and specificity of the protein samples obtained. Repeating these experiments would be costly in time and also financially. As it stands, I feel this is essential to conclusively validate their target of interest.
- Are the data and the methods presented in such a way that they can be reproduced?
The data is presented relatively well, however, it would be beneficial if deailed methods were in the main text and not in a supplementary file. Similarly, more information about the process of differentiation and how cell death/survival was quantified and validated is needed.
- Are the experiments adequately replicated and statistical analysis adequate?
In the most part yes, however there seems to be no replicates of the FLAG-out mass spec screen which is worrying given the minimal specificity observed in the current data.
Minor comments:
- Specific experimental issues that are easily addressable.
Unfortunately, the majority of experimental issues need to be addressed with more robust data which are highlighted above. However, some image analysis, quantification and classification can be amended relatively easily. For example, the live-cell imaging data should be quantified as loss of signal as discussed and RNA FISH should be used to classify XX positive cells and the XO cells can be discarded from analysis.
- Are prior studies referenced appropriately?
Most papers regarding Xist pull down and biology are discussed and referenced appropriately. However, the role in which La plays during development and its aberrant affects upon KD are seemingly downplayed. I would like to see more discussion of potential defects that could be caused due to globally altering cellular RNA folding.
- Are the text and figures clear and accurate?
For the most part, lots of the figures are clear and accurate. Apart from these exceptions.
1.The Y-axis of Figure 2D is confusing. What does 0.3 as a "sum of area" equate to? 30% of the area was ES cells? This doesn't look to be the case from Fig 2C. Also, how does the intensity of the signal compare? The area may not be a good quantification due to ES cells growing in colonies.
2.In the Movies S1-7 there are boxes around certain cells and marked with "Figure 5a - c". This seems to be incorrect as figure 5 is currently the IF staining of polycomb marks. I assume this is in relation to Figure 4b-d?
3.Similarly, in Movies S1-7, the intensities of Xist foci seem by eye to be similar. In the paper it is claimed that the Xist clouds that do form are lower in intensity. Are the Movies depicting the same range of pixel intensities? If not, this should be amended. Similarly, figure 7 seems to show relatively equivalent RNA signal at 0 h?
4.In figure 4A the data is from female XX cells, this should be highlighted to limit confusion with the male iXist data shown below in 4B-E. It would also be helpful to have the male/female icons (as in figure 3B), for each figure that has images of cells. Currently Figure 4, 5, 7, S5 and S8 are lacking these icons.
5.No explanation of the Flag-Ssb expression is given for figure S2. Furthermore, is it really necessary to express Flag-Ssb? There are reasonably good antibodies out there for Ssb as this was how it was originally found in Systemic Lupus patients. Also, no data showing the amount of Ssb being overexpressed is shown. This may have big implication to the validity of the RIP-qPCR analysis.
- Do you have suggestions that would help the authors improve the presentation of their data and conclusions?
Most of the data is presented reasonably well, but the robustness of the data somewhat retracts from their conclusions. I feel the certainty of their conclusion regarding Xist specific La binding and RNA chaperone activity is still presumptive and should be rewritten unless more robust data can confirm Xist interaction. I would also suggest deciding on the nomenclature for the protein of interest and use either La or Ssb, the continued use of both through the figures and text can get a little confusing to the reader.
Significance
- Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.
It was a good trial to use PBSb-PUFb system to purify Xist RNA binding proteins, compared to previous reports had used anti-sense oligo purification using complementary sequence to Xist RNA sequences. But currently the purification still needs further validation and repeats to confirm its use. A potential complementary technique could be to isolate Xist directly by using biotinylated probes against the PBSb sequence. The authors further claim the identification of a novel Xist RNA chaperone (La/Ssb) which they say facilitates XCI progression. This would be a novel finding in the field; however, the data is currently not robust enough to support this.
- Place the work in the context of the existing literature (provide references, where appropriate).
This work has focused on the development of a milder methodology for purifying Xist RNA during XCI. Others have published similar methodologies predominantly focusing on purifying Xist RNA directly with biotinylated probes (McHugh et al. Minaji et al and Chu et al.). Although this method boasts a milder purification method, it seems to be low yielding in Xist specific proteins. Others have shown a more robust identification of bona fide Xist binding proteins which are currently missing in this manuscript. A recent preprint from the Plath lab has identified new factors involved in XCI during differentiation and their tethering/rescue experiments are far more convincing than the ones shown in this manuscript https://www.biorxiv.org/content/10.1101/2020.03.09.979369v1. The candidate protein Ha et al have identified has multiple roles in developing cells and has shown to be important during mouse development. However, Ha et al do not robustly show that the knockdown of Ssb causes X-linked cell mortality. Alternatively, as would be presumed from Ssb's essential role in many housekeeping short non-coding RNAs, the cell death seems more ubiquitous upon shRNA KD. Therefore, the link the authors are making here are relatively weak.
- State what audience might be interested in and influenced by the reported findings.
The audience may be interested in the novel technique and the finding of a novel Xist binding protein.
- Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.
RNA biochemistry and developmental biology
