SON and SRRM2 are essential for nuclear speckle formation

  1. İbrahim Avşar Ilik
  2. Michal Malszycki
  3. Anna Katharina Lübke
  4. Claudia Schade
  5. David Meierhofer
  6. Tuğçe Aktaş

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Abstract

Nuclear speckles (NS) are among the most prominent biomolecular condensates. Despite their prevalence, research on the function of NS is virtually restricted to colocalization analyses, since an organizing core, without which NS cannot form, remains unidentified. The monoclonal antibody SC35, raised against a spliceosomal extract, is frequently used to mark NS. Unexpectedly, we found that this antibody was mischaracterized and the main target of SC35 mAb is SRRM2, a spliceosome-associated protein that sharply localizes to NS. Here we show that, the core of NS is likely formed by SON and SRRM2, since depletion of SON leads only to a partial disassembly of NS, while co-depletion of SON and SRRM2 or depletion of SON in a cell-line where intrinsically disordered regions (IDRs) of SRRM2 are genetically deleted, leads to a near-complete dissolution of NS. This work, therefore, paves the way to study the role of NS under diverse physiological and stress conditions.

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  1. Version published to 10.7554/elife.60579 on eLife
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    ##Author Response

    ###Reviewer #1:

    Major comments:

    1. The title and the conclusion that SON and SRRM2 form nuclear speckles are not supported by the data. The data show that SON and SRRM2 are necessary for nuclear speckle formation. They do not rule out that another factor is necessary, such as SRRM1, which interacts with SRRM2 and itself harbors an intrinsically-disordered domain. That is, the authors have not shown that SON and SRRM2 are also sufficient for nuclear speckle formation. Such a test is necessary to draw the strong conclusion the authors make, and precedence for such a test has been established in the study of Cajal bodies. Specifically, central factors to Cajal body formation were shown to nucleate Cajal body formation at a specific site in chromatin when such central factors were localized to that site. The authors either need to perform such a sufficiency experiment or moderate their conclusions (and title).
    1. In principle, in the immunofluorescence studies, the disappearance of mAb SC35 signal on depletion of SRRM2 does not alone prove that SRRM2 is what is visualized by the mAb SC35 in such assays. Given that this paper seeks to establish rigorously that mAb SC35 marks nuclear speckles by recognition of SRRM2, given that SRSF7 is recognized by the antibody on blots, and given that SRSF2 has been traditionally presumed the target of mAb SC35 in nuclear speckles, the rigor of this study demands that SRFS7 and SRSF2 be visualized in cells in the presence of an SRRM2 truncation to rule out that either SRSF7 or SRSF2 phenocopy SRRM2 in this assay.

    This is a valid concern and we have thought of the same principal that is if any strongly speckle-associated intrinsically disordered domain containing protein, such as SRRM1 or RBM25, two proteins that are also frequently used as NS markes, would have a similar impact on NS formation as SRRM2 has. To this end, we performed a co-depletion of SON and SRRM1 (shown in Supplementary Figure 10) in a cell line that has a TagGFP2 inserted into SRRM2 gene locus. As it can be seen from the imaging presented in this figure for 4 individual cells (but also more generally on 10 independent field imaged, (data not shown)) we did not score a reduction in the GFP intensity, or dissolution of the spherical bodies as is the case in SON-SRRM2 co-depleted cells. We observed the nuclear speckles have the round-up morphology, that is seen upon SON-KD, but are not dissolved shown with PNN staining and SRRM2-TagGFP signals. Moreover, we performed a co-depletion of RBM25 (another strongly NS-associated protein also used as a NS-marker) and SON which did not result in the dissolution of nuclear speckles (Supplementary Figure 10). Therefore, we have reached to the conclusion that SON and SRRM2 form nuclear speckles with the contribution of SON being more important for the formation and titled our study accordingly.

    Traditionally, because of the Fu & Maniatis 1992 paper, as pointed out by the reviewer, it is assumed that SC-35 recognizes SRSF2 in immunofluorescence experiments and potentially multiple SR-proteins in immunoblots. The former point, to the best of our knowledge, has never really been proven in any type of rigorous experiment. Fu lab. has generated SRSF2 K/O mice, but never provided an immunofluorescence image that shows that SC-35 signal disappears in K/O cells.

    Just to summarize our line of reasoning here:

    1. We do an unbiased IP-MS experiment, which shows that SRRM2 is the top candidate protein, at least an order of magnitude away from any other protein in the dataset by any measure. This strongly suggest that SRRM2 is the primary target of this antibody, although doesn’t prove it due to technical reasons i.e. no input normalization, some proteins produce more ‘mass-specable’ peptides than others, and larger proteins tend to produce more peptides.

    2. We carry out a biased screen of 12 SR-proteins and find that SRSF7 is strongly recognized by mAb SC-35

    3. We do IP-western blotting experiments, which correct for input and are not affected by relative ‘mass-specable’ peptide issues or protein sizes, which reveal a strong enrichment of SRRM2 (>10% of input), some enrichment for SRSF7 (~2% of input) and no enrichment for SRSF2, SRSF1 or other proteins that we have tested.

    4. Since the “35kDa” protein is so engrained with the history of this antibody and our results were most consistent with the idea that this protein is SRSF7 rather than anything else, we insert a degron tag to SRSF7. If the hypothesis is true, then we expect a shift of the SC-35 band, concomitant to the shift in SRSF7, which is indeed the case. This is not proof that SC-35 doesn’t recognize any other protein but it does provide very strong evidence (combined with the other two experiments) that the 35kDa band detected by SC-35 in immunoblots is in fact SRSF7.

    5. We then show, by TagGFP2 insertion into the SRRM2 locus, that SC-35 mAb can recognize SRRM2 specifically on immunoblots, and furthermore truncations beyond a certain point completely eliminates this signal. We also show later that siRNA mediated KD of SRRM2 also leads to the elimination of the signal from immunoblots (Supplementary Figure 9).

    6. Combining the results so far, we address the issue of immunofluorescence, i.e. which protein or proteins are responsible for this signal. We think there are two possible scenarios that could both be true based on the presented evidence so far:

    a. This signal is mainly, if not entirely, originates from SRRM2. b. The signal is a combination of SRRM2, SRSF7 and/or other SR-proteins that the SC-35 might be cross-reacting.

    1. We then take advantage of our cell lines with SRRM2 truncations. These truncated SRRM2 version are not recognized by SC-35 mAb on immunoblots, therefore it is reasonable to suspect that they will not be recognized by SC-35 mAb in immunofluorescence as well.

    2. If scenario (b) is correct and nuclear speckles are still intact in these cells (which we show that they are indeed intact, judged by SON, RBM25 and SRRM1 stainings Fig. 3A-B), then we would expect either no change in SC-35 signal, or a somewhat reduced signal. We see a complete loss of signal.

    3. Being extra careful with this result, we also mix the control cell line and SRRM2-truncated cells and image them side-by-side to address any issues related to imaging settings etc. There is no detectable SC-35 signal in truncated cells.

    4. We also show that the 35kDa band is still unchanged in SRRM2 truncated cells (Figure 2E), showing that SRSF7 itself is not affected in these cells.

    These results, combined together, show that SC-35 signal in immunofluorescence originates from SRRM2, and any other signal potentially contributed by other proteins are below the detection of immunofluorescence microscopy.

    ###Reviewer #2:

    This study reports important evidence that the widely-used SC-35 antibody primarily recognizes SRRM2 rather than the assumed SRSF2. The manuscript provides several lines of evidence supporting this conclusion, and the work has broad impact on the field of nuclear structure and function as this antibody is the most common marker for the major nuclear component, nuclear speckles.

    The one concern with the manuscript is the interpretation of some of the previous literature and understanding in the field.

    First, since the 1990s it has been widely known that the SC-35 mAb has very limited specificity for denatured proteins and was not suitable for immunoblots (see abcam page for ab11826). Indeed, the assumption has always been that it recognizes a folded epitope. Therefore, the use of western blots to conclude anything about the specificity of this antibody is inappropriate.

    Secondly, it has also been previously documented that this antibody has cross-reactivity with SRSF7 (i.e. 9G8; Lynch and Maniatis Genes Dev 1996).

    Third, most SR proteins are not abundantly observed in tryptic MS due to high cleavage of RS domains. This is particularly true of SRSF2, which has a highly "pure" RS domain (i.e. all RS repeats) that encompasses almost half of the total protein. SRRM2, on the other hand, has much more complex and degenerate RS domains that encompass a much smaller percentage of the total protein. SRRM2 is also 10x the size of SRSF2. Thus, given equal molar amounts of SRSF2 and SRRM2, one would expect at least 20x the number of peptides and much more complete coverage of SRRM2 vs. SRSF2. Therefore, while the subsequent immunoblot in Figure 1C is compelling evidence that SRRM2 is precipitated with the SC-35 antibody, while SRSF2 is not, the IP-MS data alone is not strong proof that the SC35 mAb primarily recognizes SRRM2 rather than SRSF2. The text should be revised accordingly.

    Finally, the abstract implies that the demonstration of SON as a central component of speckles is new ("elusive core"). As appropriately referenced in the text, this is not the case, rather SON is often used as a marker for nuclear speckles, and SON has long been considered to be part of the core of speckles, as knock-down has been documented by several groups to disrupt speckles. The wording in the abstract should therefore be more parsimonious.

    With all due respect to all previous researchers that have used mAb SC35 and published their results, we think that the specificity issue has become unnecessarily convoluted due to the initial inaccurate characterization. Abcam’s recommendations highlight the issue in an interesting way. In the old marketing images, abcam shows a single band in a total lysate prepared from HEK293 cells: https://www.abcam.com/ps/products/11/ab11826/reviews/images/ab11826_49518.jpg

    However, producing such an image, in our experience as we have also reported in the manuscript, is only possible under non-ideal western-blotting conditions i.e. when the transfer is not adequate to reveal proteins with large molecular weights. Intriguingly, a customer (not us) complains about an improper WB result obtained with this antibody (with a 2-star rating):

    https://www.abcam.com/sc35-antibody-sc-35-nuclear-speckle-marker-ab11826/reviews/68414?productWallTab=ShowAll

    It looks like an unexplainable high-molecular smear without the information that we provide in our manuscript, but in light of it, it’s clear that protein stained here is SRRM2.

    In our experience the antibody works perfectly fine for western blotting, and very specifically and robustly reveals SRRM2 at ~300kDa, as long as the immunoblotting conditions are optimized for large proteins. We also show that bulk of the signal around 35kDa originates from SRSF7, however as indicated by the other reviewer’s comments, and also previous research, the antibody probably cross-reacts with other proteins as well with varying degree.

    In this sense, the antibody can be used for immunoblotting, but pretty much any result obtained from such an experiment must be verified with an independent antibody or independent methods, which we did in this manuscript.

    The SC35 mAb is actually suitable for western blotting if the gel running and transfer conditions are carefully performed to have SRRM2: a) enter the gel and b) transferred properly to the membrane. Under conditions where SRRM2 is just not entering the gel (due to high percentage gels, or gels with too much bis-acrylamide), or doesn’t get transferred to a membrane (non-ideal buffer conditions, protein stuck in stacking part and cut away etc.), we have seen the unspecific bands, but we had to use the most sensitive detection reagents at hand to see those, so they are rather weak. We have provided a detailed explanation to what these conditions are in the methods section of our manuscript, but briefly: running the gel slowly allowing the protein to enter in the gel and transferring overnight with CAPS buffer were key to get the western blot working. As we have shown in Figure 2C and 2E, the majority of signal detected comes from SRRM2. The unspecific binding of SC35 mAb could only be scored if the above-mentioned conditions were not met.

    We believe what made matters historically worse has been the use Mg++ precipitation that enriches many SR proteins, but actually completely depletes SRRM2 (Blencowe et al. 1994 DOI: 10.1083/jcb.127.3.593, Figure 5, https://pubmed.ncbi.nlm.nih.gov/7962048/ ). When we’re sure that SRRM2 is in the gel though, it just shines as a single band. So in conclusion, SC-35 is reasonably specific to SRRM2, especially in immunofluorescence, but it certainly cross-reacts with other SR-proteins, especially when SRRM2 is missing for technical or biochemical reasons.

    We will update in the manuscript for the corresponding section by citing earlier studies reporting the specificity issues of mAb SC35.

    We absolutely agree that IP-MS data alone is not enough to conclude that SC-35 recognizes SRRM2, or whether it is the primary target or not. The overwhelming amount of SRRM2 peptides detected, in addition to the overwhelming amount of total peptide counts from SRRM2 does strongly suggest that it is the case, which we then followed up by IP-western blotting which controls for relative input, and the various experiments shown in later figures.

    We have looked at our MS results and found out that:

    SRSF2 was detected with 4 unique peptides with an MS/MS count of 5 and a sequence coverage of 29% (intensity 3E+07), whereas SRRM2 was detected with 227 unique peptides with an MS/MS count of 3317 and a sequence coverage of 61.9% (intensity 2E+11).

    These numbers show a 6600 times higher intensity for SRRM2 (not normalized). As the identification and abundance of different peptides/proteins can by dramatically different in MS, it is indeed correct that one should be careful with such comparisons. The only way would be to use peptide standards for both proteins and record standard curves, then a real quantitative comparison would give the true numbers. Hence, we will revise the wording of that section.

    Finally, as the reviewer has pointed out, we have not shown that speckles can be reformed by introducing ectopically expressed SON/SRRM2 into cells which now appear not to have nuclear speckles. This would indeed be the formal proof showing that SON/SRRM2 are not just necessary but also sufficient to form nuclear speckles. Such an experiment is quite challenging due to the length of these proteins and difficulty in establishing conditions where one can express these proteins, but not overexpress them which leads to round-up speckles (as shown and discussed by Belmonte lab). Therefore, we will change the title to “SON and SRRM2 are essential for the formation of nuclear speckles” to better reflect our conclusions.

    We really did try to be clear and just about the previous literature around SON. Indeed, it is clear that SON is a crucial part of NS, likely the most important component for the integrity of speckles. However, in all of these previous studies, RNAi-mediated depletion of SON, without exception, leaves behind spherical bodies that are strongly stained with mAb SC35, that also harbor other NS-markers (which we also show). This is of course not new, as we also appropriately cited previous work, however being able to dissolve these “left-over” speckles by co-depletion of SRRM2, and perhaps more importantly by deletion of the SRRM2’s C-terminal region is indeed novel.

    In essence, our results show that in the absence of SON, as shown by previous work as well, NS-associated proteins are still able to organize themselves into nuclear bodies, indicating that either all other SR-proteins without the need of another organizer clump together, or another factor (or factors) is still acting as an organizer. When we remove the C-terminus of SRRM2, which we show is the primary target of SC-35, which strongly stains these left-over nuclear bodies in the absence of SON, then deplete SON, all NS markers that we could find become diffuse, indicating that nuclear speckles no longer exist, or become too small to be detected or classified as “nuclear bodies”. Co-depletion of SON and SRRM2 leads to the same phenotype, but co-depletion of SON and SRRM1 (or RBM25) doesn’t, leaving behind spherical nuclear speckles that harbor SRRM2 which are no different than SON KD cells.

    ###Reviewer #3:

    Nuclear speckles in the last several years have attracted significant attention for their association with transcriptionally active chromosome regions (after largely being ignored by most for the previous 20 years). Overwhelmingly, a single monoclonal antibody has been used as a marker for nuclear speckles for several decades.

    This manuscript now argues convincingly that the main target that is recognized by this monoclonal antibody is not SRSF2 (SC35) as long thought, but rather SRRM2. The authors thus clarify a vast literature, while also focusing attention on the very large protein SRRM2 that in many ways resembles another nuclear speckle protein, SON. Both have huge IDRs and unusual RS repeats, while SON has been proposed to act as a scaffold for many SR-containing proteins, which is likely also true for SRRM2, by extension. Moreover, the manuscript provides a convincing explanation for why the target of this antibody was previously misidentified, by showing a lesser cross-reaction with SRSF7, of similar MW to SC35.

    Finally, the manuscript suggests that SON and SRRM2 together help nucleate nuclear speckles, as a double KD, or a SON KD in a background of a truncated SRRM2, leads to loss of nuclear speckle-like staining of other proteins normally enriched in nuclear speckles (RBM25, SRRM1, PNN). The authors go on to suggest that this double KD approach will now provide an important means of disrupting nuclear speckles to aid in functional studies.

    Interestingly, some of the results of this manuscript actually are already confirmed or consistent with previous literature. For example, a cited paper describes changes in Hi-C compartmentalization patterns after "elimination" of nuclear speckles- actually, they performed a SRRM2 KD and showed loss of SC35 staining, which is now explained as simply due to the KD that they performed. More recently, a new proteomics study of nuclear speckles (Dopie et al, JCB, 2020: https://doi.org/10.1083/jcb.201910207) reported both SON and SRRM2 as the two most highly enriched nuclear speckle proteins, with enrichment scores similar to each other but more than twice that of all other speckle proteins. Moreover, this same paper also did a SRRM2 KD and observed loss of anti-SC35 staining but not SON staining.

    Overall, I found this manuscript of significant interest for people in the nuclear cell biology field and technically thorough and well done. I just had one issue and one point to make in my main comments, plus some minor points.

    1. The evidence that nuclear speckles are nucleated by SON and SRRM2 is based on the dispersion of staining of nuclear speckle proteins RMB25, SRRM1, and PNN. However, an alternative explanation is that some other protein(s) nucleates nuclear speckles, while these other nuclear speckle proteins bind to SON and SRRM2, and are therefore enriched in nuclear speckles. To eliminate this concern, the authors could show that SON and/or SRRM2 do not bind to these proteins- for instance using co-IP or other methods. Of course, it could be that such binding or scaffolding of nuclear speckle proteins is how they form nuclear speckles. But just one protein that is not bound by SON and SRRM2 but still stains nuclear speckles after the double KD would be inconsistent with their hypothesis. Therefore, if they do find that all these proteins bind SON and/or SRRM2 they could simply discuss this as a scaffolding mechanism but qualify their conclusion based on the alternative explanation described above.
    1. In our lab we have not been comfortable using the kinase manipulations, discussed in this paper, to eliminate nuclear speckles for experimental purposes because the cells appear very sick after these manipulations. For other reasons, we also tried a double SON and SRRM2 KD. Our experience is that the cells after this double KD were also not very normal. If the authors are suggesting the SON and SRRM2 double KD as an experimental tool to disrupt nuclear speckles in order to access nuclear speckle function, then it would be valuable for them to indicate cell toxicity, etc. Many SR-protein KDs for example do not allow selection of stable cells. What about this double KD?

    The first point of Reviewer #3 has been addressed above in response to the Reviewer #2.

    We have stated that our work identifying SON and SRRM2 as the elusive core of nuclear speckles paves the way to study the nuclear speckles under physiological conditions. Here, we have used the cells 24 hours after transfection (~18 hours of knock-down) as the primary reason being that SON-KD caused a mitotic arrest if the cells were kept longer in culture. This was reported earlier in Sharma et al MBC 2010. There was no additional severity in the phenotype when the SON-KD was combined with SRRM2-KD, therefore we believe the arrest phenotype we scored is mainly due to depletion SON. In this sense, double-depletion of SON and SRRM2 can be used to study the effects of loss of NS (transcription, post-transcriptional, topological), but certainly within a time-frame of around 24 hours in cells that haven’t gone through mitosis. We will clarify this statement in the revised manuscript to avoid any misunderstanding as pointed by the reviewer. Faster depletion strategies, and/or a system where cells are mitotically arrested would be required to observe long term effects more reliably.

  3. eLife

    ###Reviewer #3:

    Nuclear speckles in the last several years have attracted significant attention for their association with transcriptionally active chromosome regions (after largely being ignored by most for the previous 20 years). Overwhelmingly, a single monoclonal antibody has been used as a marker for nuclear speckles for several decades.

    This manuscript now argues convincingly that the main target that is recognized by this monoclonal antibody is not SRSF2 (SC35) as long thought, but rather SRRM2. The authors thus clarify a vast literature, while also focusing attention on the very large protein SRRM2 that in many ways resembles another nuclear speckle protein, SON. Both have huge IDRs and unusual RS repeats, while SON has been proposed to act as a scaffold for many SR-containing proteins, which is likely also true for SRRM2, by extension. Moreover, the manuscript provides a convincing explanation for why the target of this antibody was previously misidentified, by showing a lesser cross-reaction with SRSF7, of similar MW to SC35.

    Finally, the manuscript suggests that SON and SRRM2 together help nucleate nuclear speckles, as a double KD, or a SON KD in a background of a truncated SRRM2, leads to loss of nuclear speckle-like staining of other proteins normally enriched in nuclear speckles (RBM25, SRRM1, PNN). The authors go on to suggest that this double KD approach will now provide an important means of disrupting nuclear speckles to aid in functional studies.

    Interestingly, some of the results of this manuscript actually are already confirmed or consistent with previous literature. For example, a cited paper describes changes in Hi-C compartmentalization patterns after "elimination" of nuclear speckles- actually, they performed a SRRM2 KD and showed loss of SC35 staining, which is now explained as simply due to the KD that they performed. More recently, a new proteomics study of nuclear speckles (Dopie et al, JCB, 2020: https://doi.org/10.1083/jcb.201910207 ) reported both SON and SRRM2 as the two most highly enriched nuclear speckle proteins, with enrichment scores similar to each other but more than twice that of all other speckle proteins. Moreover, this same paper also did a SRRM2 KD and observed loss of anti-SC35 staining but not SON staining.

    Overall, I found this manuscript of significant interest for people in the nuclear cell biology field and technically thorough and well done. I just had one issue and one point to make in my main comments, plus some minor points.

    1. The evidence that nuclear speckles are nucleated by SON and SRRM2 is based on the dispersion of staining of nuclear speckle proteins RMB25, SRRM1, and PNN. However, an alternative explanation is that some other protein(s) nucleates nuclear speckles, while these other nuclear speckle proteins bind to SON and SRRM2, and are therefore enriched in nuclear speckles. To eliminate this concern, the authors could show that SON and/or SRRM2 do not bind to these proteins- for instance using co-IP or other methods. Of course, it could be that such binding or scaffolding of nuclear speckle proteins is how they form nuclear speckles. But just one protein that is not bound by SON and SRRM2 but still stains nuclear speckles after the double KD would be inconsistent with their hypothesis. Therefore, if they do find that all these proteins bind SON and/or SRRM2 they could simply discuss this as a scaffolding mechanism but qualify their conclusion based on the alternative explanation described above.

    2. In our lab we have not been comfortable using the kinase manipulations, discussed in this paper, to eliminate nuclear speckles for experimental purposes because the cells appear very sick after these manipulations. For other reasons, we also tried a double SON and SRRM2 KD. Our experience is that the cells after this double KD were also not very normal. If the authors are suggesting the SON and SRRM2 double KD as an experimental tool to disrupt nuclear speckles in order to access nuclear speckle function, then it would be valuable for them to indicate cell toxicity, etc. Many SR-protein KDs for example do not allow selection of stable cells. What about this double KD?

  4. eLife

    ###Reviewer #2:

    This study reports important evidence that the widely-used SC-35 antibody primarily recognizes SRRM2 rather than the assumed SRSF2. The manuscript provides several lines of evidence supporting this conclusion, and the work has broad impact on the field of nuclear structure and function as this antibody is the most common marker for the major nuclear component, nuclear speckles.

    The one concern with the manuscript is the interpretation of some of the previous literature and understanding in the field.

    First, since the 1990s it has been widely known that the SC-35 mAb has very limited specificity for denatured proteins and was not suitable for immunoblots (see abcam page for ab11826). Indeed, the assumption has always been that it recognizes a folded epitope. Therefore, the use of western blots to conclude anything about the specificity of this antibody is inappropriate.

    Secondly, it has also been previously documented that this antibody has cross-reactivity with SRSF7 (i.e. 9G8; Lynch and Maniatis Genes Dev 1996).

    Third, most SR proteins are not abundantly observed in tryptic MS due to high cleavage of RS domains. This is particularly true of SRSF2, which has a highly "pure" RS domain (i.e. all RS repeats) that encompasses almost half of the total protein. SRRM2, on the other hand, has much more complex and degenerate RS domains that encompass a much smaller percentage of the total protein. SRRM2 is also 10x the size of SRSF2. Thus, given equal molar amounts of SRSF2 and SRRM2, one would expect at least 20x the number of peptides and much more complete coverage of SRRM2 vs. SRSF2. Therefore, while the subsequent immunoblot in Figure 1C is compelling evidence that SRRM2 is precipitated with the SC-35 antibody, while SRSF2 is not, the IP-MS data alone is not strong proof that the SC35 mAb primarily recognizes SRRM2 rather than SRSF2. The text should be revised accordingly.

    Finally, the abstract implies that the demonstration of SON as a central component of speckles is new ("elusive core"). As appropriately referenced in the text, this is not the case, rather SON is often used as a marker for nuclear speckles, and SON has long been considered to be part of the core of speckles, as knock-down has been documented by several groups to disrupt speckles. The wording in the abstract should therefore be more parsimonious.

  5. eLife

    ###Reviewer #1:

    Major comments:

    1. The title and the conclusion that SON and SRRM2 form nuclear speckles are not supported by the data. The data show that SON and SRRM2 are necessary for nuclear speckle formation. They do not rule out that another factor is necessary, such as SRRM1, which interacts with SRRM2 and itself harbors an intrinsically-disordered domain. That is, the authors have not shown that SON and SRRM2 are also sufficient for nuclear speckle formation. Such a test is necessary to draw the strong conclusion the authors make, and precedence for such a test has been established in the study of Cajal bodies. Specifically, central factors to Cajal body formation were shown to nucleate Cajal body formation at a specific site in chromatin when such central factors were localized to that site. The authors either need to perform such a sufficiency experiment or moderate their conclusions (and title).

    2. In principle, in the immunofluorescence studies, the disappearance of mAb SC35 signal on depletion of SRRM2 does not alone prove that SRRM2 is what is visualized by the mAb SC35 in such assays. Given that this paper seeks to establish rigorously that mAb SC35 marks nuclear speckles by recognition of SRRM2, given that SRSF7 is recognized by the antibody on blots, and given that SRSF2 has been traditionally presumed the target of mAb SC35 in nuclear speckles, the rigor of this study demands that SRFS7 and SRSF2 be visualized in cells in the presence of an SRRM2 truncation to rule out that either SRSF7 or SRSF2 phenocopy SRRM2 in this assay.

  6. eLife

    ##Preprint Review

    This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript.

    ###Summary:

    This study has yielded two significant contributions. First, the study recharacterized a widely used antibody, mAb SC35, which was initially raised against the spliceosome and characterized both as targeting the 35 kDa protein, SRSF2, an intensely studied splicing regulatory factor, and as marking nuclear speckles, which in the last several years have attracted significant attention for their association with transcriptionally active chromosome regions (after largely being ignored by most for the previous 20 years). The authors present a series of rigorously designed and carefully carried out experiments demonstrating that the 35 kDa factor that mAb recognizes is instead SRSF7. Moreover, the authors present compelling evidence that the primary target of mAb SC35 is a ~300 kDa protein, SRRM2, a spliceosomal factor originally discovered as a nuclear matrix factor and later defined as a nuclear speckle component. In the most convincing experiments establishing these targets the authors show that mAb SC35 signals shift, when the molecular weight of SRSF7 or SRRM2 is varied, and that the signal disappears when SRSF7 is depleted. Given the use of mAb SC35 for nearly three decades, these results suggest that tens if not hundreds of papers require re-interpretation. This study reminds us again of the necessity of rigorous validation of antibodies.

    Second, the authors investigate the role of SRRM2 in the formation of nuclear speckles. Previous studies have shown that knock down of the nuclear speckle factor SON leads to a compaction of nuclear speckles but not their entire dissolution, implicating a role for at least one additional factor in nuclear speckle formation; other studies have implicated an array of factors as being required for nuclear speckle formation. Here, the authors show that truncation or knock down of SRRM2, in contrast to several other nuclear speckles factors, also reduce nuclear speckle number, although more modestly than SON, and the truncation or knockdown of SRRM2 in combination with the depletion of SON reduces nuclear speckles more than SON depletion alone. The authors interpret these findings to indicate that SON and SRRM2, both of which harbor intrinsically-disordered domains, form nuclear speckles in human cells, as the title indicates. Further, the authors suggest that the double knockdown provides a new tool to study nuclear speckle function. Overall, this study provides surprising and important insight into a commonly used mAb and valuable new perspectives on nuclear speckles, which have the potential to transform future studies. The study will be of broad interest to those interested in splicing, nuclear speckles, antibody specificity, and more generally, liquid-liquid phase separation.

  7. Version published to 10.1101/2020.06.19.160762v1 on bioRxiv