SON and SRRM2 are essential for nuclear speckle formation

1Nuclear speckles (NS) are membraneless nuclear bodies in the interchromatin-space of the nucleus that contain high concentrations of RNA-processing and some transcription factors but are devoid of DNA. Under normal conditions, they appear as irregularly shaped, dynamic structures that show hallmarks of phase-separated condensates, such as fusion and deformation under pressure in living cells. Despite their prevalence, the function of NS remains largely unknown, although they have been proposed to act as reservoirs for splicing factors, and association with NS have been shown to correlate with enhanced transcription and RNA-processing. NS have been shown to be involved in replication of herpes simplex virus, processing and trafficking of Influenza A virus mRNA, detaining repetitive RNA originating from the transcription of repeat expanded loci that trigger Huntington's disease, spinocerebellar ataxia and dentatorubral–pallidoluysian atrophy, but also repetitive RNA from artificial constructs that produce RNA capable of phase-separation in vitro. Studying the role of NS involves visualizing them with a fluorescently tagged factor that localizes to NS, or the use of antibodies that show specific staining of NS. Similar to nucleoli and other membraneless bodies of the nucleus, NS disassemble during early stages of mitosis, and assemble back following telophase. Several protein kinases are thought to be involved in this process, such as DYRK3, chemical inhibition of which leads to aberrant phase-separation. Overexpression of DYRK3, or CLK1 on the other hand leads to dissolution of NS in interphase cells, underscoring the importance of phosphorylation in NS integrity. Unlike several other biomolecular condensates, a specific core necessary for NS formation has not yet been identified, and it has been hypothesized that stochastic self-assembly of NS-associated factors could lead to the formation of NS. One of the most frequently used reagents to locate NS is the monoclonal antibody SC35, which was raised against biochemically purified spliceosomes, and reported to be an antibody against SRSF2. Testament to the importance of this antibody, NS are also referred to as ‘SC35 domains’. Although, the name SC35 and SRSF2 are used synonymously and to annotate orthologues of SRSF2 not only in mammalian species but also in species such as D. melanogaster and A. thaliana, mAb SC35 is reported to cross-react with SRSF1, and potentially with other SR-proteins as well. Furthermore, fluorescently tagged SRSF2 shows staining patterns incompatible with mAb SC35 stainings under identical experimental conditions. Intrigued by these inconsistencies, we undertook a systemic re-characterization of the mAb SC35 and its cellular targets.

2In order to characterize the cellular targets of the SC35 mAb, we carried out an Immunoprecipitation Mass-Spectrometry (IP-MS) experiment. Whole-cell extracts prepared from HAP1 cells were used to immunoprecipitate endogenous targets SC35 mAb, with a matched IgG mAb serving as a control. The immunoprecipitated proteins were then analyzed by mass-spectrometry (see Methods for details). In total, we identified 432 proteins that were significantly enriched in the SC35 purifications compared to controls (p<0.05, at least two peptides detected in each biological replicate). Surprisingly, the most enriched protein in the dataset, both in terms of unique peptides detected, total intensities and MS/MS spectra analyzed, is neither SRSF2 nor one of the canonical SR-proteins, but a high-molecular weight RNA-binding protein called SRRM2 (Figure 1A, Figure 1—figure supplement 1A), an NS-associated protein with multiple RS-repeats. Analysis of the top 108 targets, corresponding to the third quartile, using the STRING database shows a clear enrichment for the spliceosome and NS (Figure 1—figure supplement 1B), validating the experimental approach. We were also able to detect all SR-proteins in our dataset, however their scores are dwarfed by that of SRRM2’s (Figure 1—figure supplement 1C). Thus, contrary to initial expectations, the IP-MS results strongly suggest but do not prove that SC35 mAb primarily recognizes SRRM2, at least in the context of an immunoprecipitation experiment.

3Before exploring SRRM2 as a potential mAb SC35 target protein, we decided to first take an unbiased look at SR-proteins and the ability of mAb SC35 to recognize them. To this end, we cloned all 12 canonical SR-proteins in humans into an expression plasmid, and created stable-cell lines expressing these proteins under the control of a doxycycline-inducible promoter (Figure 1—figure supplement 2). We used a biotin-acceptor peptide as a tag, and carried out stringent purifications using streptavidin beads to exclude non-specific co-purification of unrelated SR-proteins, and examined the eluates using immunoblotting. Surprisingly, our results show that the main target of mAb SC35 on these immunoblots is SRSF7 (Figure 1B), which, when untagged, runs at approximately ~35 kDa on polyacrylamide gels similar to SRSF2 (the tag combination that we use shifts both proteins by ~15 kDa). To exclude any artefacts that could originate from the use of tagged proteins, we used whole-cell extracts from HEK293 cells and immunoprecipitated targets of mAb SC35 and analyzed the eluates via immunoblotting. Consistent with the results of the IP-MS experiment, and tagged-SRSF1-12 purifications, we observed a very clear enrichment for SRRM2 and SRSF7, but not for SRSF2, SRSF1 or other factors (Figure 1C). In order to determine whether the 35 kDa band recognized by mAb SC35 in immunoblots of cellular lysates is composed of multiple proteins, in addition to SRSF7, we created a cell line where we inserted the FKBP12F36V degron tag homozygously into the C-terminus of SRSF7 in HEK293 cells. Even without any treatment, it is evident that the 35 kDa band robustly recognized by mAb SC35 in wild-type cells completely disappears in SRSF7FKBP12 cells (Figure 1D, compare lanes 1 and 2 with 3 and 4), and a new band around ~50 kDa, where the FKBP12F36V-tagged SRSF7 runs, emerges (Figure 1D, arrowhead). Treatment of these cells with 1 µM of dTAG7 for 6 hr lead to the depletion FKBP12F36V-tagged SRSF7, and to the depletion of the newly-emerged ~50 kDa protein recognized by mAb SC35. The identity of a fainter band around 37 kDa, which is insensitive to tagging SRSF7 or dTAG treatment remains unknown. These results strongly suggest that the 35 kDa namesake protein revealed by SC35 mAb on immunoblots is SRSF7 and any contribution to this signal from other proteins is negligible to none.

4Even though our results show that SC35 mAb specifically recognizes SRSF7 rather than SRSF2, both proteins have significant nucleoplasmic pools in addition to their localization to NS which is not easily reconciled with the immunofluorescence stainings obtained with the SC35 mAb that are virtually restricted to NS. Intriguingly, SRRM2, which is by far the most enriched protein in our immunoprecipitations with mAb SC35, is a relatively large (~300 kDa) protein, that readily co-purifies with spliceosomes, shows liquid-like behaviour in cells and co-localizes near-perfectly with mAb SC35-stained NS. In addition, SRRM2 and its yeast counterpart Cwc21/Cwf21 is located in the recent cryo-EM structures of the spliceosome, where it joins the spliceosome at the Bact stage where it seems to support the activated conformation of PRP8’s switch-loop both in humans and yeast. Predating the recent cryo-EM structures by almost a decade, the yeast orthologue of SRRM2, Cwf21p, has been shown to directly interact with Prp8p (PRPF8) and Snu114p (EFTUD2) which are also among the most enriched proteins in our mAb SC35 immunoprecipitations (; Figure 1—figure supplement 1A). Furthermore, a more recent tandem-affinity purification of the protist Trypanosoma orthologue of SRRM2 revealed Prp8, U5-200K (SNRNP200, also known as Brr2), U5-116K (EFTUD2, also known as Snu114) and U5-40K (SNRNP40) as major interaction partners.

5Taking into consideration the fact the mAb SC35 was raised against biochemically purified spliceosomes, together with the aforementioned observations in the scientific literature and our IP-MS results which identified SRRM2 as the top target, we hypothesize that mAb SC35 was most likely raised against SRRM2, and it recognizes SRRM2 in most if not all immunological assays that utilizes mAb SC35 where SRRM2 is not depleted or unintentionally omitted due to technical reasons.

6In order to test the veracity of this claim, we designed a series of experiments in human cells. Since, to our knowledge, SC35 mAb has not been shown to recognize SRRM2 on immunoblots, we first created tagged and truncated SRRM2 constructs in living cells. To this end, we generated 11 cell lines that remove between 4 and 2322 amino acids from the SRRM2 protein (full-length: 2752 a.a., numbering from Q9UQ35-1) by inserting a TagGFP2 (referred to as GFP for simplicity) sequence followed by an SV40 polyadenylation signal into 11 positions of the SRRM2 gene in HAP1 cells using a CRISPR/Cas9-based technique called CRISPaint (; Figure 2A). The deepest truncation removes 84% of SRRM2, which includes almost all its IDRs, together with two regions enriched for serine and arginine residues, leaving behind 13 RS-dipeptides out of a total of 173 (Figure 2—figure supplement 1A,B). The GFP-tagged, in vivo truncated proteins (referred to as truncations 0 to 10, shortened as tr0 - tr10, Figure 2B) are then immunoprecipitated using GFP-trap beads and the eluates were analyzed by immunoblotting. This experiment shows that SC35 mAb indeed recognizes SRRM2 on immunoblots (Figure 2C, lane 2). Interestingly, the signal from SC35 mAb remains relatively stable up until SRRM2tr4 which removes 868 a.a. from the SRRM2 C-terminus, the signal appears to be reduced in SRRM2tr5 which removes 1014 a.a. and becomes completely undetectable from SRRM2tr6 onward (Figure 2C and Figure 2—figure supplement 1C). The same blot was stripped and re-probed with a polyclonal antibody raised against the N-terminus of SRRM2, common to all truncations, which show that SRRM2 is detectable throughout, and thus indicating that the epitope(s) recognized by mAb SC35 reside between amino acids 1,360–1884 of SRRM2.

7In order to assess the efficiency and the specificity of the GFP-pull-down, we used a wild-type lysate without any GFP insertion, together with lysates made from SRRM2tr0 and SRRM2tr10 cells, which served as the negative control, positive control and the deepest truncation (tr10) we generated, respectively. The immunoblot with mAb SC35 once again clearly shows that near-full-length SRRM2tr0 is recognized by mAb SC35 to the same extent as the SRRM2 polyclonal antibody, while SRRM2tr10 is not detected by mAb SC35 at all but strongly with SRRM2 polyclonal antibody (Figure 2D). These blots also show that full-length SRRM2 co-purifies SRRM1 and to a lesser extent RBM25, while both interactions are severely compromised in SRRM2tr10. Furthermore, the absence of any signal in SRRM2tr10 input lane probed with mAb SC35 (Figure 2D, top left lane 3), and the emergence of a shorter ~100 kDa protein in the complete absence of a ~300 kDa signal in the SRRM2 blot (Figure 2D, bottom left, lane 3) shows that SRRM2tr10 cells have a homozygous insertion of the GFP construct, which was also confirmed by genotyping PCR (Figure 2—figure supplement 1D). This result further indicates that the large IDRs of SRRM2 are not essential for cell viability, at least in HAP1 cells.

8These results can be puzzling, since we first show that mAb SC35 specifically recognizes a 35 kDa band which we reveal to be SRSF7 (Figure 1), but later, in a separate set of experiments, we also show that mAb SC35 specifically recognizes a ~300 kDa band, which we reveal to be SRRM2 (Figure 2), while the original study describing mAb SC35 reports a single 35 kDa band recognized by mAb SC35 on immunoblots. The solution to this conundrum presented itself in the form of altering the immunoblotting technique. Using whole-cell extracts prepared from wild-type cells, together with SRRM2tr0 and SRRM2tr10 cells, in a gel system where we can interrogate both small and large proteins simultaneously, we were able to detect both SRRM2 and SRSF7 on the same blot (Figure 2E). These blots prove that the ~300 kDa band is indeed SRRM2, since it completely disappears in SRRM2tr10 lysates (which is accompanied by the appearance of a ~100 kDa band in SRRM2 blots) while the much fainter 35 kDa band corresponding to SRSF7 (Figure 1) remains unaltered.

9These experiments provide strong support for our hypothesis that the main target of SC35 mAb is SRRM2, a protein proven to be part of spliceosomes, against which this antibody was raised, and suggests that a cross-reactivity towards SRSF7, likely in combination with immunoblotting techniques not suitable to detect large proteins, obscured this fact for more than two decades.

10mAb SC35 is typically used as an antibody in immunofluorescence experiments that reveals the location of NS in mammalian cells. In light of the evidence presented here, it can be assumed that mAb SC35 primarily stains SRRM2 in immunofluorescence stainings, as in immunoblotting experiments. In order to test if this is indeed the case, we took advantage of the SRRM2tr10 cells. These cells are viable and express a severely truncated SRRM2 that is not recognized by mAb SC35 on immunoblots (Figure 2).

11SRRM2tr10 cells, together with SRRM2tr0 cells serving as a control, were stained with antibodies against various nuclear speckle markers, including mAb SC35 (Figure 3A). These results show that SC35 signal virtually disappears in SRRM2tr10 cells, while other markers of NS, such as SON, SRRM1, and RBM25 appear unaltered, ruling out a general defect in NS (Figure 3A). As an additional control, we also mixed SRRM2tr10 cells with SRRM2tr0 cells together before formaldehyde fixation, and repeated the antibody stainings, in order to be able to image these two cell populations side-by-side. SRRM2tr10 and SRRM2tr0 cells are easily distinguished from each other since the latter show a typical nuclear speckle staining whereas the former has a more diffuse, lower intensity GFP signal. These images clearly show that mAb SC35, obtained from two separate vendors, no longer stains NS or any other structure in SRRM2tr10 cells, whereas other NS markers, including SRSF7, appear unaltered (Figure 3B,C, Figure 3—figure supplement 1).

12Taken together, our results show that mAb SC35, which was raised against a spliceosomal extract, was most likely raised against SRRM2, a ~300 kDa protein that, unlike SRSF2 or SRSF7 is present in spliceosomes of both in yeast and humans. We show that mAb SC35 directly recognizes SRRM2 between amino acids 1,360–1,884, and that the main signal from mAb SC35 corresponds to SRRM2 both in immunoblots and immunofluorescence images. It is important to note that these results were obtained from unsynchronised human cells, which are mostly at the interphase stage of the cell-cycle. mAb SC35 might recognize additional targets in mitotic cells or cells derived from non-mammalian species.

13It is also interesting to note that this is not the first time an antibody is serendipitously raised against SRRM2 and was later discovered to recognize SRRM2 only after the fact: In 1994, reported three murine monoclonal antibodies, B1C8, H1B2, and B4A11 which were raised against nuclear matrix preparations. All three antibodies showed extensive co-localization with NS, although a co-localization between mAb SC35 and B4A11 could not directly be assessed since both mAb SC35 and B4A11 are reported to be IgG mAbs. In a separate work, showed that B4A11 is an antibody against SRRM2, suggesting that SRRM2 is present both in spliceosomal purifications and nuclear matrix preparations.

14During this work, we noticed the remarkable size difference between human SRRM2 protein (2752 a.a.), and its unicellular counterparts S. cerevisiae Cwc21 (133 a.a), S. pombe Cwf21 (293 a.a) and T. Brucei U5-Cwc21 (143 a.a). Moreover, while all three proteins share a conserved N-terminus, which interact with the spliceosome, the serine and arginine-rich extended C-terminus of human SRRM2 is predicted to be completely disordered (Figure 2—figure supplement 1B). Intrigued by this observation, we compiled all metazoan protein sequences of SRRM2, together with SRRM1, RBM25, PNN, SON, PRPF8 and COILIN, and analyzed their size distributions (Figure 4A). This analysis confirmed that, unlike SRRM1, RBM25, PNN, PRPF8 or coilin, SRRM2 indeed has a very broad size distribution within metazoa (Figure 4—figure supplements 1–2). Strikingly, SON follows this trend with orthologues as small as 610 a.a in the basal metazoan sponge A. queenslandica, and as large as 5561 a.a in the frog X. tropicalis. Increase in protein size appears to involve IDR extensions, especially for SRRM2, but also for SON (Figure 4B, Figure 4—figure supplements 1–2), suggesting a role in LLPS-mediated condensate formation, which was shown to be the case for both SRRM2 and SON in living cells.

15Putting together the observation that places SON and SRRM2 at the center of NS (, with the interpretation that SC35 stains SRRM2 in their microscopy work), the presence of SRRM2 at the center of collapsed speckles in SON knock-down experiments, and the peculiar variation in the sizes of SON and SRRM2 during evolution involving gain of IDRs, we hypothesize that SON, together with SRRM2 are essential for NS formation, such that SRRM2 continues to serve as a platform for NS-associated proteins in SON-depleted cells.

16In order to test this hypothesis, we used the SRRM2tr0 and SRRM2tr10 cells as a model, which allowed us to simultaneously detect SON, SRRM2 and an additional NS marker in the same cell. We chose SRRM1, which is used as a marker for NS in immunofluorescence experiments and located at IGCs in electron microscopy experiments (; RBM25, which is one of two recommended factors to mark NS by the Human Protein Atlas; ‘’ n.d.; ) (the other being SRRM2), localizes to NS through its RE/RD-rich mixed-charge domain that was recently shown to target proteins to NS and PNN, which localizes to NS in human cells.

17As reported previously, depletion of SON leads to collapsed speckles in SRRM2tr0 cells, with SRRM2, SRRM1, PNN, and RBM25 localizing to these spherical NS to different extents (Figure 5A, Figure 5B, Figure 5—figure supplement 3, compare SRRM2tr0 cells, control vs SON siRNA treatment). In SRRM2tr10 cells on the other hand, where the truncated SRRM2 has a significant nucleoplasmic pool already in control siRNA treated cells, depletion of SON leads to a near-complete diffusion of truncated SRRM2, which is followed by RBM25 (Figure 5B), SRRM1 (Figure 5—figure supplement 3A) and PNN. Using ilastik and CellProfiler, we quantified the signal detected in NS, and compared it to signal detected in the entire nucleus for each cell in every condition for each protein investigated (Figure 5—figure supplement 2). These results show that truncated SRRM2 shows reduced NS localization (Figure 5C, right), while RBM25, SRRM1, and PNN are localized at NS to a similar extent in SRRM2tr0 and SRRM2tr10 cells, although with a broader distribution in SRRM2tr10 cells. Depletion of SON in SRRM2tr0 leads to a significant reduction in NS localization for all proteins, verifying SON’s importance for NS formation. Depletion of SON in SRRM2tr10 cells, however, leads to a more dramatic loss of NS localization for all proteins (Figure 5 and Figure 5—figure supplement 3), underscoring the essential role of SRRM2’s extended IDR in the formation of NS, especially in SON-depleted cells. Number of Cajal bodies, determined by COILIN staining, remains unaltered in all conditions (Figure 5—figure supplement 3B).

18Next, to independently verify these observations, we knocked-down SON and SRRM2, individually and simultaneously in HEK293 cells where we endogenously tagged SRRM2 with TagGFP2 at the C-terminus with the same reagents used to create SRRM2tr0 HAP1 cells. Similar to the HAP1 model, depletion of SON alone leads to collapsed NS where SRRM2, RBM25, PNN, and SRRM1 localize to spherical NS to some extent but with a significant non-NS pool in the nucleus (Figure 5—figure supplement 4). Depletion of SRRM2 alone also leads to delocalization of PNN, SRRM1, and RBM25 from NS, but not to the extent seen with SON depletion. Co-depletion of SON and SRRM2 leads to near-complete delocalization of all proteins investigated, mirroring the results obtained from the HAP1 model (Figure 5—figure supplement 4A,B,C and D). These results cannot be explained by reduced protein stabilities, as none of the proteins except for SON and SRRM2 show significant changes in their amounts as judged by immunoblotting (Figure 5—figure supplement 4E). Finally, co-depletion of SON together with SRRM1 or RBM25 does not lead to diffusion of spherical NS marked by SRRM2, indicating that SRRM2 has a unique role in NS formation and cannot be substituted by other NS-associated factors (Figure 5—figure supplement 5).

19Our results have broad implications with respect to the biology in and around NS. One of the primary culprits in the so-called ‘reproducibility crisis’ in natural sciences is considered to be mischaracterized antibodies, which led to initiatives to validate them appropriately, and large consortia such as ENCODE publishes specific guidelines for the characterization antibodies that are used to generate data pertinent to the ENCODE project (‘,’ n.d.).

20It is therefore reasonable to suspect that the dissonance between observations made with mAb SC35 and subsequent observations made with SRSF2 reagents could have led to misinterpretation of vast amounts of primary data. It is beyond the scope of this work to review each and every study that has used this antibody in the last 30 years, however, since both SRSF2 and SRRM2 are clinically important genes, and SRRM2 gene is highly to intolerant to loss-of-function mutations in human populations (expected/observed ratio for SRRM2 is 6%, median expected/observed ratio for all gene variants is 48%), the opportunity cost incurred when results generated with mAb SC35 have been misinterpreted must be carefully considered.

21For example, GSK-3, a protein kinase implicated in Alzheimer's disease, has been shown to phosphorylate SRSF2. This study reported that inhibition of GSK-3 leads to spherical NS in cortical neurons, which was interpreted as SRSF2 accumulating at NS upon GSK-3 inhibition. Based on this assumption, a cerebral cortex lysate was immunoprecipitated with mAb SC35, and a 35 kDa band was shown to be phosphorylated by recombinant GSK-3, whereas neither SRSF7 nor SRRM2 were pursued as potential candidates. Interestingly, a more recent study reported that SRRM2 is a target of GSK-3. In another study, mAb SC35 has been used as a reporter for phosphorylated SRSF2 in immunohistochemical analysis of tissue samples obtained from patients with Non Small Cell Lung Carcinoma, and was reported to be upregulated in cancer tissues compared to normal tissues. SRRM2's potential role in these syndromes therefore remains unexplored.

22In a more non-clinical setting, recent microscopy work suggests that RNA polymerase II, through its intrinsically disordered CTD, switches from transcriptional condensates to NS during progression from initiation to productive elongation, which is in line with recent work that links NS to augmented gene expression. In this particular work, initial observations made with the SC35 mAb have been followed by fluorescently tagged SRSF2 protein for live-cell imaging analysis and condensates formed by SRSF2 are used as a surrogate for splicing condensates in vitro. Furthermore, ChIP-seq profiles generated with mAb SC35 were interpreted as SRSF2 occupancy in vivo, which was shown to be enriched at the 3’-ends of genes, together with serine-2 phosphorylated RNAPII. SRRM2’s potential role in these processes remains unknown. In another study, loss of mAb SC35 signal in cells treated with siRNAs against SRRM2 has been interpreted to be a sign of NS disassembly, and based on this knowledge, a more recent study interrogated the 3D conformation of the mouse genome in SRRM2-depleted cells using Hi-C, results of which are interpreted to be a consequence of NS disruption. A recent super resolution microscopy study made use of the mAb SC35 to show that SRSF2 is at the core of NS together with SON, and remains at the core after depletion of SON. In this study, a minimalist computational model was used to model the distribution of five NS-associated factors (SON, SRSF2 on account of SC35 mAb stainings, MALAT1, U1, and U2B'') which could have benefited from the knowledge that SC35 mAb stains SRRM2 rather than SRSF2, considering that pairwise interactions between SRRM2 and other components would be drastically different to SRSF2's potential interactions due to the extensive IDRs of SRRM2 compared to SRSF2. This study also highlights the fact that a crucial aspect of nuclear speckle biology, namely whether NS are nucleated by specific factors or not, remained an open question.

23In contrast, many membraneless organelles have been shown to depend on a small number of factors which act as scaffolds or nucleation points for their formation. Paraspeckles for instance require lncRNA NEAT1, without which paraspeckles do not form, Cajal bodies are disrupted or disappear in the absence of COILIN, SMN, FAM118B or WRAP53, and PML bodies are nucleated by PML. In the same vein, SRRM2 has been suggested to be essential for the formation of NS, however, this idea was based on the disappearance of mAb SC35 signal in cells transfected with siRNAs against SRRM2, which is the expected result taking the evidence presented here into account, but does not prove that SRRM2 is essential or important for NS formation. Other candidates that were put forward as essential or important for the formation of NS include lncRNA MALAT1, SRSF1, PNN, and SON, all of which, with the exception of PNN, lead to the formation of ‘collapsed’ speckles rather than a bulk release of NS-associated factors and their diffusion into the nucleoplasm, which would indicate a true loss of NS. Depletion of PNN was shown to either lead to ‘collapsed’ speckles or to loss of NS altogether, but under conditions that also lead to degradation of all SR-proteins tested in that particular study. To our knowledge, NS could only be successfully dissolved by overexpression of CLK1/STY kinase, which phosphorylates SR-proteins, DYRK3, another protein kinase that can dissolve multiple membraneless bodies, overexpression of PPIG, a peptidyl-proline isomerase or more recently by overexpression of TNPO3, which is an import factor that binds to phosphorylated SR-residues. Such observations and lack of evidence to the contrary, led to the idea that NS formation happens through stochastic self-assembly of NS-associated factors, without the need for an organizing core. In this work, we show that the spherical bodies left over after depletion of SON, which are strongly stained with mAb SC35, and also by other NS markers such as SRRM1, PNN, RBM25 can be dissolved by either co-depleting SRRM2 together with SON, or depleting SON in a cell-line where we deleted the intrinsically disordered C-terminus of SRRM2. Co-depletion of SON together with either SRRM1 or RBM25, two relatively large proteins that also possess prominent intrinsically disordered regions and localize to NS, does not lead to the dissolution of left-over NS, which remain as spherical bodies strongly stained with SRRM2. Since SON is essential for mitosis, conditions reported here therefore open an approximately 24 hr window to study the transcriptional and post-transcriptional effects of lacking NS in human cells.

24Taken together, our results show that a widely-used monoclonal antibody to mark NS, SC35 mAb, was most likely raised against SRRM2 and not against SRSF2 as it was initially reported. We speculate that this mischaracterization hindered the identification of the core of NS, without which NS do not form, which we show to consist most likely of SON and SRRM2. We found that these two factors, unlike other splicing related proteins analyzed, have gone through a remarkable length extension through evolution of metazoa over the last ~0.6–1.2 billion years, mostly within their IDRs which are typically involved in LLPS and formation of biomolecular condensates. The exact mechanism of NS formation by SON together with SRRM2, and the evolutionary forces that led to the dramatic changes in their lengths remain to be discovered.

25Flp-In T-REx HEK293 (Thermo Fisher Scientific Catalog Number: R78007) cells were cultured according to manufacturer’s recommendations. The cells were cultured in DMEM with Glutamax supplemented with Na-Pyruvate and High Glucose (Thermo Fisher Scientific Catalog Number: 31966–021) in the presence of 10% FBS (Thermo Fisher Scientific Catalog Number: 10270106) and Penicillin/Streptomycin (Thermo Fisher Scientific Catalog Number: 15140–122). Before the introduction of the transgenes cells were cultured with a final concentration of 100 µg/mL zeocin (Thermo Fisher Scientific Catalog Number: R250-01) and 15 µg/mL blasticidin (Thermo Fisher Scientific Catalog Number: A1113903). To generate the stable cell lines pOG44 (Thermo Fisher Scientific Catalog Number: V600520) was co-transfected with pcDNA5/FRT/TO (Thermo Fisher Scientific Catalog Number: V652020) containing the gene of interest (GOI are SRSF1 to 12 in this case) in a 9:1 ratio. Cells were transfected with Lipofectamine 2000 (Thermo Fisher Scientific Catalog Number: 11668019) on a 6-well plate format with total 1 µg DNA (i.e. 900 ng of pOG44 and 100 ng of pcDNA5/FRT/TO+GOI) according to the transfection protocol provided by the manufacturer. 24 hr after the transfection cells were split on 3 wells of a 6-well plate at 1:6, 2:6 and 3:6 dilution ratios to allow efficient selection of Hygromycin B (Thermo Fisher Scientific Catalog Number: 10687010). The Hygromycin selection was started at the 48 hr after transfection time point with a final concentration of 150 µg/mL and refreshed every 3–4 days until the control non-transfected cells on a separate plate were completely dead (takes approximately 3 weeks from the start of transfection until the cells are expanded and frozen). Induction of the transgene was done over-night with a final concentration of 0.1 µg/mL doxycycline. The cells were validated by performing immunofluorescence by FLAG antibody and western blotting of nuclear and cytoplasmic fractions.

26Human HAP1 parental control cell line was purchased from Horizon (Catalog Number: C631) and cultured according to the instructions provided by the manufacturer. The cells were cultured in IMDM (Thermo Fisher Scientific Catalog Number: 12440–053) in the presence of 10% FBS (Thermo Fisher Scientific Catalog Number: 10270106) and Penicillin/Streptomycin (Thermo Fisher Scientific Catalog Number: 15140–122). See Supplementary file 1 for the list of sgRNAs used in generation of cell lines with CRISPaint. Cells were co-transfected with three plasmids according to the CRISPaint protocol. Cas9 and sgRNA are provided by same plasmid in 0.5 µg final amount, Frame selector plasmid (depending on the cut site selector 0, +1 or +2 had to be chosen) is also in 0.5 µg final amount, the TagGFP2_CRISPaint plasmid was provided at a 1 µg final amount. Therefore the total 2 µg DNA was transfected into cells on 6-well plate format using Lipofectamine 2000. 24 hr after the transfection the cells were expanded on 10 cm culture plates to allow efficient Puromycin (Thermo Fisher Scientific Catalog Number: A1113803) selection. The Puromycin selection is provided in the tag construct and is driven by the expression from the gene locus (in this case the human SRRM2 gene locus). Puromycin selection was started at 48 hr after transfection at 1 µg/mL final concentration and was refreshed every 2 days and in total was kept for 6 days. After the colonies grew to a visible size the colonies were picked by the aid of fluorescence microscope EVOS M5000. PCR screening of the colonies was performed using genotyping oligos listed in Supplementary file 1 using Quick Extract DNA Extraction Solution (Lucigen Catalog Number: QE09050) according to manufacturer’s protocol in a PCR machine and DreamTaq Green Polymerase (Thermo Fisher Scientific Catalog Number: K1081) using 58°C annealing temperature and 1 min extension time.

27SRSF7-FKBP12F36V knock-in cells were generated in HEK293T cells (ordered from ATCC, CRL3216 and cultured according to the protocol provided) by co-transfecting the sgRNA, Frame selector and mini-circle constructs prepared according to the CRISPaint protocol using Lipofectamine 2000 on a 6-well plate format. This time we used two separate tag donor plasmids to increase the chances of obtaining homozygous clones. The constructs were identical except for the selection antibiotic. Cells are expanded on 10 cm culture plates 24 hr after transfection. At 48 hr after transfection the double selection was initiated. One allele was selected by Puromycin at 1 µg/mL final concentration, whereas the other allele was selected by blasticidin at 15 µg/mL final concentration for 6 days in total. After the removal of selection cells were kept on the same plate until there were big enough colonies. Colonies were picked under a sterile workbench and screened for homozygosity using western blotting with SRSF7 antibody. The degradation of tagged-SRSF7 was induced by adding dTAG7 reagent at a final 1 µM concentration and keeping for 6 hr.

28SRRM2tr0-GFP Flp-In TREx HEK293 cells were generated using the same strategy as described above for HAP1 cells. Upon Puromycin selection cells were used as a pool (without sorting or colony picking) in immunofluorescence experiments.

29Cell lines are regularly checked for the absence of Mycoplasma using a PCR based detection kit (Jena Biosciences PP-401).

30Prior to the seeding of cells, the round glass 12 mm coverslips are coated with poly-L-Lysine hydrobromide (Sigma P9155) for HEK293 cells. The coating is not necessary for the imaging of HAP1 cells. For 1 day of knock-down 40,000 cells are plated on coverslips placed into the wells of 24-well plates on the day before the siRNA transfections. Pre-designed silencer select siRNA (Ambion) are ordered for SRRM2 (ID: s24004), SON (ID: s13278), SRRM1 (ID: s20020) and RBM25 (ID: s33912). Negative control #1 of the silencer select was used for control experiments. 5 nM (for double transfections) or 10 nM (for single transfections) of each siRNA is forward transfected using Lipofectamine RNAiMAX Reagent (Thermo Fisher Scientific Catalog Number: 13778075) according to manufacturer’s instructions. The cells were fixed for imaging 24 hr after transfection.

31Cells on coverslips were washed with PBS and crosslinked with 4% paraformaldehyde in PBS (Santa Cruz Biotechnology, sc-281692) for 10 min at room temperature, and washed three times with PBS afterwards. Permeabilization was carried out with 0.5% Triton-X in PBS, 10 min at RT. Cells were washed twice with 0.1% Triton-X in PBS and blocked with 3% BSA (constituted from powder BSA, Roche Fraction V, sold by Sigma Catalog Number: 10735078001) in PBS for 30 min at RT. Primary antibodies were diluted in 3% BSA in PBS, and cells were incubated with diluted primary antibodies for ~16 hr at 4°C in a humidified chamber. Cells were then washed three times with 0.1% Triton-X in PBS and incubated with fluorescently labelled secondary antibodies, diluted 1:500 in 3% BSA for 1 hr at RT, and washed three times with 0.1% Triton-X in PBS. To counterstain DNA, cells were incubated with Hoechst 33258 (1µg/mL, final) for 5 min at RT, and washed once with PBS. Coverslips are briefly rinsed with distilled water and mounted on glass slides using Fluoromount-G (SouthernBiotech, 0100–01) and after a few hours, sealed with CoverGrip (Biotium, #23005) and left in a dark chamber overnight before imaging.

32COIL (Cell Signaling Technology, D2L3J, #14168), FLAG-M2 (Sigma, F3165), PNN (Abcam, ab244250), RBM25 (Sigma, HPA070713-100UL), SC-35 (Santa Cruz Biotechnology, sc-53518), SC-35 (Sigma, S4045), SON (polyclonal rabbit, Sigma, HPA023535), SON (monoclonal mouse, Santa Cruz sc-398508), SRRM1 (Abcam, ab221061), SRRM2 (Thermo Fisher Scientific, PA5-66827).

33Images were acquired with a Zeiss LSM880 microscope equipped with an AiryScan detector, using the AiryScan Fast mode with the Plan-Apochromat 63x/1.40 Oil DIC M27 objective. The dimensions of each image were 134 x 134 x 4 µm (Width x Height x Depth), 20 z-stacks were acquired for each image with a step size of 200 nm. Maximum Intensity Projections were created using Zen software (Zeiss) and used for further analysis.

34Nuclear speckle identification, segmentation and intensity calculations were carried out using ilastik and CellProfiler. Briefly, eight images were used to train a model that demarcates NS using ilastik (v.1.3.3post2). CellProfiler was then used to segment nuclei and NS using the probability maps created for each image by ilastik. The data was then analyzed in a Jupyter Lab environment using pandas, SciPy, NumPy and plotted with matplotlib and seaborn. Raw imaging data, models used to train the images, CellProfiler pipelines, and Jupyter notebooks are available.

35Pierce MS-Compatible Magnetic IP Kit (Protein A/G) (Thermo Fisher Scientific, Catalog Number: 90409) was used to prepare samples for mass-spectrometry according manufacturer’s instructions, where approximately 15 million HAP1 cells (~80% confluent 15 cm dishes) were used per IP. Briefly, HAP1 cells were trypsinized, washed with ice-cold PBS and re-suspended with 500 µL of ‘IP-MS Cell Lysis Buffer’ which was supplemented with 1x cOmplete Protease Inhibitor Cocktail (Roche, 11697498001) and 1x PhosSTOP (Roche, 4906845001). Cells were then homogenized using a Bioruptor Plus sonifier (30 s ON, 30 s OFF, five cycles on HI). Remaining cellular debris was removed by centrifugation at 21.130 rcf for 10 min at 4°C, supernatants were transferred to fresh tubes. 2.5 µL of SC35 mAb (Sigma-Aldrich, S4045) and 25 µL of control IgG1 (Santa Cruz, sc-3877) was used for the SC35 and control IP samples (3 each), respectively and immune-complexes are allowed to form overnight (~16 hr) in the cold-room (~6°C) with end-to-end rotation. Next morning, lysates were incubated with 25 µL of Protein A/G beads for 1 hr in the cold-room, the beads were then washed with 500 µL of ice-cold 50 mM Tris.Cl pH 7.4, 100 mM NaCl, 0.1% Tween-20. The beads were resuspended with the same buffer supplemented with RNaseI (Ambion, AM2295, final concentration 0.02 U/µL) and incubated at 37°C for 5 min. The beads were then washed three times with ‘Wash A (+10 mM MgCl2)’ and twice with ‘Wash B’ buffer.

36The buffer for the three SC35 samples and controls was exchanged with 100 µL of 50 mM NH4HCO3. This was followed by a tryptic digest including reduction and alkylation of the cysteines. Therefore, the reduction was performed by adding tris(2-carboxyethyl)phosphine with a final concentration of 5.5 mM at 37°C on a rocking platform (500 rpm) for 30 min. For alkylation, chloroacetamide was added with a final concentration of 24 mM at room temperature on a rocking platform (500 rpm) for 30 min. Then, proteins were digested with 200 ng trypsin (Roche, Basel, Switzerland) shaking at 600 rpm at 37°C for 17 hr. Samples were acidified by adding 2.2 µL 100% formic acid, centrifuged shortly, and placed on the magnetic rack. The supernatants, containing the digested peptides, were transferred to a new low protein binding tube. Peptide desalting was performed according to the manufacturer’s instructions (Pierce C18 Tips, Thermo Scientific, Waltham, MA). Eluates were lyophilized and reconstituted in 11 µL of 5% acetonitrile and 2% formic acid in water, briefly vortexed, and sonicated in a water bath for 30 s prior injection to nano-LC-MS/MS.

37LC-MS/MS was carried out by nanoflow reverse-phase liquid chromatography (Dionex Ultimate 3000, Thermo Scientific) coupled online to a Q-Exactive HF Orbitrap mass spectrometer (Thermo Scientific), as reported previously. Briefly, the LC separation was performed using a PicoFrit analytical column (75 μm ID ×50 cm long, 15 µm Tip ID; New Objectives, Woburn, MA) in-house packed with 3 µm C18 resin (Reprosil-AQ Pur, Dr. Maisch, Ammerbuch, Germany). Peptides were eluted using a gradient from 3.8% to 38% solvent B in solvent A over 120 min at 266 nL per minute flow rate. Solvent A was 0.1% formic acid and solvent B was 79.9% acetonitrile, 20% H2O, 0.1% formic acid. Nanoelectrospray was generated by applying 3.5 kV. A cycle of one full Fourier transformation scan mass spectrum (300–1750 m/z, resolution of 60,000 at m/z 200, automatic gain control (AGC) target 1 × 106) was followed by 12 data-dependent MS/MS scans (resolution of 30,000, AGC target 5 × 105) with a normalized collision energy of 25 eV. To avoid repeated sequencing of the same peptides, a dynamic exclusion window of 30 s was used.

38Raw MS data were processed with MaxQuant software (v1.6.0.1) and searched against the human proteome database UniProtKB with 21,074 entries, released in December 2018. Parameters of MaxQuant database searching were a false discovery rate (FDR) of 0.01 for proteins and peptides, a minimum peptide length of seven amino acids, a first search mass tolerance for peptides of 20 ppm and a main search tolerance of 4.5 ppm. A maximum of two missed cleavages was allowed for the tryptic digest. Cysteine carbamidomethylation was set as a fixed modification, while N-terminal acetylation and methionine oxidation were set as variable modifications. The MaxQuant processed output files can be found in Supplementary file 2, showing peptide and protein identification, accession numbers, % sequence coverage of the protein, and q-values.

39The mass-spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD021814.

40Streptavidin-pulldowns (Figure 1B) were carried out using stable-cell-line expression SRSF1-12 proteins. Briefly, for each cell line, ~1 million cells (one well of a 6-well dish, ~90% confluent) were induced with 0.1 µg/mL doxycycline (final) for ~16 hr, solubilised with 500 µL of 1xNLB (1X PBS, 0.3M NaCl, 1% Triton X-100, 0.1% TWEEN 20) + 1x PhosSTOP, sonicated with Bioruptor (30 s ON/OFF, five cycles on LO) and centrifuged for 10 min at ~20.000 rcf at 4°C to remove cellular debris. Biotinylated target proteins were purified with 25 µL (slurry) of MyONE-C1 streptavidin beads (Thermo Fisher Scientific, 65002), pre-washed with 1x NLB + 1x PhosSTOP, for 2 hr in the cold-room with end-to-end rotation. Beads were washed 3 times with 500 µL of 1x NLB (5 min each), bound proteins were eluted with 50 µL of 1xLDS sample buffer (Thermo Fisher Scientific, NP0007) + 100 mM beta-mercaptoethanol at 95°C for 5 min. Eluates were loaded on a 4–12% Bis-Tris gel (Thermo Fisher Scientific, NP0322PK2) and transferred to a 0.45 µm PVDF membrane (Merck Millipore, IPVH00010) with 10 mM CAPS (pH 11) + 10% MeOH, for 900 min at 20V. Primary antibodies were used at a dilution of 1:1000 in SuperBlock (Thermo Fisher Scientific, 37515). Membranes were incubated with the diluted primaries overnight in the cold-room.

41SC35 and IgG immunoprecipitations (Figure 1C) were carried out using a whole-cell extract prepared from wild-type HEK293 cells. Briefly, ~10 million cells were resuspended with 600 µL of 1x NLB + 1x cOmplete Protease Inhibitor Cocktail + 1x PhosSTOP, and kept on ice for 15 min. The lysate was cleared by centrifugation at ~20.000 rcf for 10 min at 4°C. Clarified lysate was split into two tubes; to one tube 25 µL of control IgG1 (Santa Cruz, sc-3877) was added, to the other 2.5 µL of SC35 mAb (Sigma-Aldrich, S4045), immune-complexes are allowed to form for 3 hr in the cold-room with end-to-end rotation. 40 µL of Protein G Dynabeads (Thermo Fisher Scientific, 10003D, washed and resuspended with 200 µL of 1x NLB + PI + PS) was used to pull-down target proteins. Beads were washed three times with 1xNLB, briefly with HSB (50 mM Tris.Cl pH 7.4, 1M NaCl, 1% IGEPAL CA-630, 0.1% SDS, 1 mM EDTA) and finally with NDB (50 mM Tris.Cl pH 7.4, 0.1M NaCl, 0.1% TWEEN 20). Bound proteins were eluted with 50 µL of 1xLDS sample buffer (Thermo Fisher Scientific, NP0007) + 100 mM beta-mercaptoethanol at 80°C for 10 min. Immunoblotting was carried out as described for streptavidin pull-downs, except transfer was carried out with 25 mM Tris, 192 mM glycine, 20% (v/v) for 90 min at 90V in the cold-room.

42Pull-down of truncated SRRM2 proteins (Figure 2C–E) were carried out using whole-cell lysate prepared from respective HAP1 cell lines. The protocol is essentially identical to SC35 and IgG immunoprecipitations described above, with these notable differences: (1) For pull-downs, 25 µL (slurry) of GFP-trap agarose beads were used (Chromotek, gta), incubations were carried out overnight in the cold-room (2) For Figure 2C and Figures 2D, 3-8% Tris-Acetate gels (Thermo Fisher Scientific, EA0375PK2) were used, for Figure 2E a 4–12% Bis-Tris gel was used (3) Gels were run at 80V for 3 hr (4) Transfers were carried out with 10 mM CAPS (pH 11) + 10% MeOH for 900 min at 20V.

43ADAR1(Santa Cruz Biotechnology, sc-73408), alpha-Tubulin (Santa Cruz Biotechnology, sc-32293), COIL (Cell Signaling Technology, D2L3J, #14168), DHX9 (Abcam, ab183731), FLAG-M2 (Sigma, F3165), GFP (Chromotek, PAGB1), Myc-tag (Cell Signaling Technology, 9B11, #2276), Phospho-Rpb1 CTD (Ser2) (Cell Signaling Technology, E1Z3G, #13499), PNN (Abcam, ab244250), RBM25 (Sigma, HPA070713-100UL), SC-35 (Sigma, S4045), SRSF1 (SF2/ASF, Santa Cruz Biotechnology, sc-33652), SON (polyclonal rabbit, Sigma, HPA023535), SRRM1 (Abcam, ab221061), SRRM2 (Thermo Fisher Scientific, PA5-66827), SRSF2 (Abcam, ab28428), SRSF7 (MBL, RN079PW), U1-70K (Santa Cruz Biotechnology, sc-390899), U2AF65 (Santa Cruz Biotechnology, sc-53942).

44There are many commercially available antibodies that are labeled as ‘SC35’, however only some of them are actually clones of the original SC-35 antibody reported by Fu and Maniatis in 1990. These are: s4045 from Sigma-Aldrich, sc-53518 from Santa Cruz Biotechnology and ab11826 from Abcam. Some antibodies are sold as ‘SC35’ antibodies, but they are antibodies specifically raised against SRSF2. These are: ab204916 and ab28428 from Abcam and 04–1550 from Merck (can be found with the clone number 1SC-4F11). Neither list is exhaustive.

45Unless indicated otherwise, all data analysis tasks were performed using Python 3.7 with scientific libraries Biopython, pandas, NumPy, matplotlib and seaborn. Code in the form of Jupyter Notebooks is available in GitHub repository: https://github.molgen.mpg.de/malszycki/SON_SRRM2_speckles.

46Vertebrate SRRM2, SON, PRPF8, SRRM1, RBM25, Pinin, and Coilin orthologous protein datasets were downloaded from NCBI’s orthologs and supplemented with orthologues predicted for invertebrate species. For this purpose, OrthoFinder was used on a set of Uniprot Reference Proteomes. Invertebrate orthologues were then mapped to NCBI RefSeq to remove fragmentary and redundant sequences. The resulting dataset can be accessed here: https://doi.org/10.5281/zenodo.4065244 and was manually curated to remove evident artefacts lacking conserved domains or displaying striking differences from closely related sequences. Protein lengths were plotted using the seaborn package and descriptive statistics calculated using the pandas package.

47In order to resolve phylogenetic relationships between species contained in SRRM2 and SON datasets, organism names were mapped to the TimeTree database. Disorder probability was predicted using IUPred2A and MobiDB-Lite and plotted as a heatmap using matplotlib.