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Alternative splicing of the SRRM2 targets FES and MUC1 regulates innate immunity and cell homeostasis. (A) SRRM2 regulates alternative splicing of FES. Map of reads: IGV software; PSI: rMATS analysis; Splicing validation by RT-PCR (Figure S5A). (B) Diagram of mouse and human FES domains including SH2 affected by alternative splicing in human only, and targeting shRNAs (green arrow showing shRNA targeting iso1/2, red arrow showing shRNA targeting all isoforms). Iso, isoform. (C) Isoform-specific knockdown effect of FES shRNAs. (D) Relative expression of TNFα and IL1β proinflammatory cytokines upon isoform-specific knockdown of FES in human macrophage-like THP-1 cells under LPS (1 ng/ml) for 6 h. To observe a stronger phenotype upon FES knockdown, we used a weak LPS stimulation with little effect on THP-1 inflammation. (E) Sunburst diagram showing the effect of CPT drug, and combined CPT plus SRRM2 knockdown on SRRM2 DASEs. (F) Cell viability analysis of THP-1 cells upon SRRM2 knockdown with either CPT or AZA treatment for 24 h. (G) Expression of MUC1 upon SRRM2 knockdown in THP-1, measured by RNA-seq or qRT-PCR. (H) RNA-seq analysis showing the reads and annotations of spliced exons of MUC1 upon SRRM2 knockdown and CPT treatment. (I) RT-PCR validation of the skipping of exons 3 or 4 upon SRRM2 knockdown. (J) PSI differences and statistical analysis of MUC1 exon 3 and 4 upon SRRM2 knockdown and combined treatment with CPT. (K) Graphic diagram showing SRRM2 regulates exon 4 skipping to modulate MUC1 shedding. MUC-N: N-terminal domain; MUC1-C: C-terminal domain; SEA: Sperm protein, Enterokinase and Agrin; GSVVV: cleavage site for proteases TACE or ADAM9; Glycosylation: protein modification. Shedding generates soluble MUC1, promoting cancer progression or regulating immune response. Data are mean ± s.e.m. (n = 3 experiments). Statistical significance determined using one-way ANOVA or two-way ANOVA (F) with Bonferroni correction for multiple comparison (*P < 0.05; **P < 0.01; ***P < 0.001. ns: no significant difference).
Source publication
SRRM2 is a nuclear-speckle marker containing multiple disordered domains, whose dysfunction is associated with several human diseases. Using mainly EGFP-SRRM2 knock-in HEK293T cells, we show that SRRM2 forms biomolecular condensates satisfying most hallmarks of liquid-liquid phase separation, including spherical shape, dynamic rearrangement, coales...
Contexts in source publication
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... introns both upstream and downstream of cassette exons skipped by SRRM2 knockdown were on average 5.5 kb shorter than the introns with increased inclusion upon knockdown, and control introns as well ( Figure 5C). Motif analysis using MEME (37) revealed that the cassette exons skipped by SRRM2-knockdown are enriched with the GGUGG motif either in the intronic or exonic regions (Fig- ure S6A). Overall, these analyses reveal the specific features exhibited by SRRM2-regulated DASEs ( Figure 5D). ...
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... test the effects of SRRM2 in co-transcriptional splicing, we treated THP-1 cells with two drugs that slow down Pol II transcription, 5,6-dichloro-1--D-ribofuranosylbenzimidazole (DRB) and camptothecin (CPT) (38,39). RNA-seq global assay showed that SRRM2 knockdown enhanced the effect of CPT/DRB induced alternative splicing change on SRRM2 targets ( Figure S6B), indicating that SRRM2 tends to affect splicing in the same direction as processive Pol II. ...
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... we investigated the functional impact of the SRRM2-regulated alternative splicing. The FES SH2 domain encoded by cassette exon 10 is essential for attenuating the TLR signaling pathway ( Figure S7A) (41,42), and this exon was significantly excluded upon SRRM2 knockdown in either monocytic or macrophage-like THP-1 cells ( Figure 6A and S5D). We further tested the isoform-specific function of FES using shRNA targeting either long isoforms only (with exon 10) or both short and long isoforms ( Figure 6B (57). ...
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... FES SH2 domain encoded by cassette exon 10 is essential for attenuating the TLR signaling pathway ( Figure S7A) (41,42), and this exon was significantly excluded upon SRRM2 knockdown in either monocytic or macrophage-like THP-1 cells ( Figure 6A and S5D). We further tested the isoform-specific function of FES using shRNA targeting either long isoforms only (with exon 10) or both short and long isoforms ( Figure 6B (57). (C) Volcano plot showing log 2 fold change (log 2 FC) of RNA expression of splicing factors between knockdown and control in THP-1. ...
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... regulation of G0 to G1 transition Regulation of G0 to G1 transition on PMA-induced macrophage-like THP-1. We found that the TNFα and IL1β pro-inflammatory cytokines were significantly upregulated upon specific knockdown of all isoforms and SH2-containing FES isoforms ( Figure 6D). The shRNA targeting all isoforms more strongly upregulated these cytokines, possibly due to its higher knockdown efficiency than the exon-10 specific shRNA ( Figure 6C), which could not be further optimized due to the limited length of exon 10. ...
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... found that the TNFα and IL1β pro-inflammatory cytokines were significantly upregulated upon specific knockdown of all isoforms and SH2-containing FES isoforms ( Figure 6D). The shRNA targeting all isoforms more strongly upregulated these cytokines, possibly due to its higher knockdown efficiency than the exon-10 specific shRNA ( Figure 6C), which could not be further optimized due to the limited length of exon 10. Hence, the exon-10 containing FES isoforms contribute to the observed transcriptional modulation of proinflammatory cytokines in macrophages by SRRM2 ( Figure S7B). ...
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... the exon-10 containing FES isoforms contribute to the observed transcriptional modulation of proinflammatory cytokines in macrophages by SRRM2 ( Figure S7B). This may partially explain why human immune cells are more sensitive to LPS, as mice only have the FES isoform with SH2 domain, while human macrophages exhibit significant skipping of exon 10 ( Figure 6B). Collectively, these data suggest that SRRM2 attenuates innate inflammatory responses via FES, although there might be other factors and pathways involved. ...
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... GO analysis shows that CPT treatment slows down Pol II and induces cell cycle arrest and programmed cell death ( Figure S7C). SRRM2 knockdown enhanced the CPT drug treatment effect on SRRM2 DASEs ( Figure 6E). Consistent with this, when we combined SRRM2 knockdown with either CPT or azacytidine (AZA) treatment, we found that THP-1 cell viability was significantly lower upon combination treatment compared to drug alone ( Figure 6F). ...
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... knockdown enhanced the CPT drug treatment effect on SRRM2 DASEs ( Figure 6E). Consistent with this, when we combined SRRM2 knockdown with either CPT or azacytidine (AZA) treatment, we found that THP-1 cell viability was significantly lower upon combination treatment compared to drug alone ( Figure 6F). The DEGs or DASEs of CPT/SRRM2 knockdown compared to CPT alone were enriched in the pathways of cell cycle arrest and DNA damage ( Figure S7D). ...
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... the DASE gene list, the oncoprotein MUC1 (Mucin-1) is a potential AML therapeutic target (44). Here we show that SRRM2 knockdown significantly downregulated MUC1 at both mRNA and cell surface expression level ( Figure 6G, S7E and F). SRRM2 knockdown significantly induced strong skipping of either exon 3 or exon 4 in THP-1 cells ( Figure 6H and I), with the combination treatment inducing more skipping of exon 4 compared to either SRRM2 knockdown or CPT alone (Fig- ure 6J). ...
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... we show that SRRM2 knockdown significantly downregulated MUC1 at both mRNA and cell surface expression level ( Figure 6G, S7E and F). SRRM2 knockdown significantly induced strong skipping of either exon 3 or exon 4 in THP-1 cells ( Figure 6H and I), with the combination treatment inducing more skipping of exon 4 compared to either SRRM2 knockdown or CPT alone (Fig- ure 6J). Disruption of the GSVVV cleavage site in MUC1 SEA (Sperm protein, Enterokinase and Agrin) domain encoded by exon 4 prevents shedding of MUC1 mediated by proteases such as TACE or ADAM9, and MUC1 shedding is a mechanism to promote cancer progression, as well as it regulates innate immune response (45,46) ( Figure 6K and S7F). ...
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... we show that SRRM2 knockdown significantly downregulated MUC1 at both mRNA and cell surface expression level ( Figure 6G, S7E and F). SRRM2 knockdown significantly induced strong skipping of either exon 3 or exon 4 in THP-1 cells ( Figure 6H and I), with the combination treatment inducing more skipping of exon 4 compared to either SRRM2 knockdown or CPT alone (Fig- ure 6J). Disruption of the GSVVV cleavage site in MUC1 SEA (Sperm protein, Enterokinase and Agrin) domain encoded by exon 4 prevents shedding of MUC1 mediated by proteases such as TACE or ADAM9, and MUC1 shedding is a mechanism to promote cancer progression, as well as it regulates innate immune response (45,46) ( Figure 6K and S7F). ...
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... knockdown significantly induced strong skipping of either exon 3 or exon 4 in THP-1 cells ( Figure 6H and I), with the combination treatment inducing more skipping of exon 4 compared to either SRRM2 knockdown or CPT alone (Fig- ure 6J). Disruption of the GSVVV cleavage site in MUC1 SEA (Sperm protein, Enterokinase and Agrin) domain encoded by exon 4 prevents shedding of MUC1 mediated by proteases such as TACE or ADAM9, and MUC1 shedding is a mechanism to promote cancer progression, as well as it regulates innate immune response (45,46) ( Figure 6K and S7F). Thus, the targeting MUC1 by SRRM2 contributes to the reduction of cell viability by AML drugs. ...
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Citations
... HNRNPU, also called SAF-A (for Scaffold Attachment Factor A), is a DNA-and RNA-binding protein that was identified as a constituent of the nuclear matrix capable of binding to nuclear matrix/scaffold-attachment regions (S/MAR; Kiledjian and Dreyfuss, 1992;Romig et al., 1992;Jenke et al., 2002Jenke et al., , 2004. More recent studies indicate that HNRNPU is a chromatin scaffolding protein which, by oligomerizing with chromatin-associated RNA (caRNA), controls chromatin architecture and cellular gene expression (Nozawa et al., 2017;Fan et al., 2018;Xu et al., 2022). SRRM2, a member of the SR-related protein family, is involved in pre-mRNA maturation as a catalytic component of the spliceosome together with SRRM1 (Blencowe et al., 2000). ...
... SRRM2, a member of the SR-related protein family, is involved in pre-mRNA maturation as a catalytic component of the spliceosome together with SRRM1 (Blencowe et al., 2000). Recent investigations also indicated that SRRM2, is a major component of nuclear speckles (Ilik et al., 2020;Xu et al., 2022). Both proteins, in particular SRRM2, are phosphorylated on multiple sites. ...
... Initially described as a component of the spliceosome and a member of the SR family of proteins, SRRM2 was recently identified as a main scaffold of nuclear speckles (Ilik et al., 2020). In particular, SRRM2 can form liquid condensates in a kinase-controlled fashion (Rai et al., 2018;Xu et al., 2022). This RBP was also up-phosphorylated in HIV-1-infected cells, in which it regulated alternative splicing of viral RNAs, as well as following infection of human macrophages with influenza A virus (Wojcechowskyj et al., 2013;Soderholm et al., 2016). ...
Phosphorylation is a major post-translation modification (PTM) of proteins which is finely tuned by the activity of several hundred kinases and phosphatases. It controls most if not all cellular pathways including anti-viral responses. Accordingly, viruses often induce important changes in the phosphorylation of host factors that can either promote or counteract viral replication. Among more than 500 kinases constituting the human kinome only few have been described as important for the hepatitis B virus (HBV) infectious cycle, and most of them intervene during early or late infectious steps by phosphorylating the viral Core (HBc) protein. In addition, little is known on the consequences of HBV infection on the activity of cellular kinases. The objective of this study was to investigate the global impact of HBV infection on the cellular phosphorylation landscape early after infection. For this, primary human hepatocytes (PHHs) were challenged or not with HBV, and a mass spectrometry (MS)-based quantitative phosphoproteomic analysis was conducted 2- and 7-days post-infection. The results indicated that while, as expected, HBV infection only minimally modified the cell proteome, significant changes were observed in the phosphorylation state of several host proteins at both time points. Gene enrichment and ontology analyses of up- and down-phosphorylated proteins revealed common and distinct signatures induced by infection. In particular, HBV infection resulted in up-phosphorylation of proteins involved in DNA damage signaling and repair, RNA metabolism, in particular splicing, and cytoplasmic cell-signaling. Down-phosphorylated proteins were mostly involved in cell signaling and communication. Validation studies carried out on selected up-phosphorylated proteins, revealed that HBV infection induced a DNA damage response characterized by the appearance of 53BP1 foci, the inactivation of which by siRNA increased cccDNA levels. In addition, among up-phosphorylated RNA binding proteins (RBPs), SRRM2, a major scaffold of nuclear speckles behaved as an antiviral factor. In accordance with these findings, kinase prediction analysis indicated that HBV infection upregulates the activity of major kinases involved in DNA repair. These results strongly suggest that HBV infection triggers an intrinsic anti-viral response involving DNA repair factors and RBPs that contribute to reduce HBV replication in cell culture models.
... In previous studies, changes of SRRM2 variants were reported to be closely related with Parkinson's disease [56,57]. Although the role of SRRM2 alternative splicing has yet been uncovered in cancers, its aberrant expression and germline mutation was found to contribute to thyroid cancer progression and recurrence [58,59]. The DMKN gene, which harbors five splice variants (α, β, γ, δ, and ε), has been previously studied to suggest that suppressing DMKN-β/γ reduces the invasiveness and migratory capabilities of pancreatic cancer cells, potentially impacting the epithelial-mesenchymal transformation (EMT). ...
Background
SRSF1, a member of Serine/Arginine-Rich Splicing Factors (SRSFs), has been observed to significantly influence cancer progression. However, the precise role of SRSF1 in osteosarcoma (OS) remains unclear. This study aims to investigate the functions of SRSF1 and its underlying mechanism in OS.
Methods
SRSF1 expression level in OS was evaluated on the TCGA dataset, TAGET-OS database. qRT-PCR and Western blotting were employed to assess SRSF1 expression in human OS cell lines as well as the interfered ectopic expression states. The effect of SRSF1 on cell migration, invasion, proliferation, and apoptosis of OS cells were measured by transwell assay and flow cytometry. RNA sequence and bioinformatic analyses were conducted to elucidate the targeted genes, relevant biological pathways, and alternative splicing (AS) events regulated by SRSF1.
Results
SRSF1 expression was consistently upregulated in both OS samples and OS cell lines. Diminishing SRSF1 resulted in reduced proliferation, migration, and invasion and increased apoptosis in OS cells while overexpressing SRSF1 led to enhanced growth, migration, invasion, and decreased apoptosis. Mechanistically, Gene Ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, and Gene Set Enrichment Analysis (GSEA) revealed that the biological functions of SRSF1 were closely associated with the dysregulation of the protein targeting processes, location of the cytosolic ribosome, extracellular matrix (ECM), and proteinaceous extracellular matrix, along with the PI3K-AKT pathway, Wnt pathway, and HIPPO pathway. Transcriptome analysis identified AS events modulated by SRSF1, especially (Skipped Exon) SE events and (Mutually exclusive Exons) MXE events, revealing potential roles of targeted molecules in mRNA surveillance, RNA degradation, and RNA transport during OS development. qRT-PCR confirmed that SRSF1 knockdown resulted in the occurrence of alternative splicing of SRRM2, DMKN, and SCAT1 in OS.
Conclusions
Our results highlight the oncogenic role of high SRSF1 expression in promoting OS progression, and further explore the potential mechanisms of action. The significant involvement of SRSF1 in OS development suggests its potential utility as a therapeutic target in OS.
... Both protein and RNA molecules can phase separate into biomolecular condensates that regulate cellular activities by organizing biochemical reactions 1,[7][8][9] . Co-assembled ribonucleoprotein (RNP) granules play critical roles in RNA metabolism 2 such as transcription [10][11][12] , splicing [12][13][14] , translation 15,16 , and decay 17,18 under both physiological and pathological conditions 9 . Proteins often serve as scaffold molecules that drive the phase separation, and RNAs usually partition into a condensate as guest molecules 8,19,20 . ...
Protein-rich biomolecular condensates formed by phase separation1 play a crucial role in cellular RNA regulation by the selective recruitment and processing of RNA molecules. The functional impact of condensates on RNA biology relies on the residence time of individual RNA molecules within a condensate, which is governed by intra-condensate diffusion - the slower or more confined the diffusion, the longer the residence time. However, the spatiotemporal organization of RNA and protein diffusion within a single condensate remains largely unknown due to the challenge of accurately profiling intra-condensate diffusion behaviors down to the single-molecule level. Here we introduced a general condensate-tethering approach that allows single molecule tracking (SMT) of fluorescently labeled proteins and RNAs within non-wetted spherical 3D condensates without the interference of condensate motions. We found that a significant fraction of RNA and protein molecules are locally confined, rather than freely diffusive, within a model condensate formed by full-length, tag-free, RNA-binding protein Fused-in-Sarcoma (FUS), known for its critical roles in RNA biology under both physiological and pathological conditions. Dynamic Point Accumulation for Imaging in Nanoscale Topography (PAINT) reconstruction further revealed that RNA and proteins are confined to distinct slow-moving nanometer-scale regions, termed nanodomains, within a single condensate. Remarkably, nanodomains affect the diffusion but not the density of the confined biomolecules, supporting local percolation rather than a secondary phase separation within the condensate as their origin. Beyond their regulatory roles on RNA residence time, nanodomains engender both elevated local connectivity and altered chemical environment, a prerequisite for pathological liquid-to-solid transition of FUS during aging. In summary, our study uncovers a patterned spatial organization of both protein and RNA molecules within a single condensate, revealing distinct diffusion dynamics that affect molecular retention time and interactions underlying cellular function and pathology.
... In addition, nuclear speckles were demonstrated to promote co-transcriptional splicing through increased binding of spliceosomes to pre-mRNAs from speckle-proximal genes (Bhat et al., 2023;Ding and Elowitz, 2019). In addition to promoting constitutive splicing, nuclear speckles were also demonstrated to facilitate splicing of stress-related genes under ribotoxic stress in a regulated fashion (Sung et al., 2023), as well as impacting alternative splicing (Xu et al., 2022). However, within these proposed functions of nuclear speckle in splicing, fundamental questions are not addressed: (1) Do all genes require speckles for co-or post-transcriptional splicing? ...
Nuclear speckles, a type of membraneless nuclear organelle in higher eukaryotic cells, play a vital role in gene expression regulation. Using the reverse transcription-based RNA-binding protein binding sites sequencing (ARTR-seq) method, we study human transcripts associated with nuclear speckles. We identify three gene groups whose transcripts demonstrate different speckle localization properties and dynamics: stably enriched in nuclear speckles, transiently enriched in speckles at the pre-mRNA stage, and not enriched in speckles. Specifically, we find that stably-enriched transcripts contain inefficiently spliced introns. We show that nuclear speckles specifically facilitate splicing of speckle-enriched transcripts. We further reveal RNA sequence features contributing to transcript speckle localization, underscoring a tight interplay between genome organization, RNA cis-elements, and transcript speckle enrichment, and connecting transcript speckle localization with splicing efficiency. Finally, we show that speckles can act as hubs for the regulated retention of introns during cellular stress. Collectively, our data highlight a role of nuclear speckles in both co- and post-transcriptional splicing regulation.
... Human SRRM2 (also known as SRm300) exhibits multiple stretches of serines and arginines ( Fig. S4C), localizes to nuclear speckles 63,64 , and upholds the nuclear speckle structure together with the RNA-binding protein SON 52 . SRRM2 is a splicing factor involved in pre-mRNA splicing as part of the spliceosome B act stage 65 , but it is not required for constitutive pre-mRNA splicing 66 . ...
... The alternative splicing (AS) analysis revealed hundreds of unique alternative splicing events in cells forming TFIID complexes associating with or lacking SRRM2. These results are in line with reports showing that SRRM2 is not required for constitutive splicing 66 and that it regulates splicing of only a subset of RNAs coding for proteins involved in innate immunity, cell homeostasis 64,66 or inflammatory metabolism 87 . Further, and expectedly, the observed changes in AS upon reduced TFIID-SRRM2 complex formation were less pronounced preprint (which was not certified by peer review) is the author/funder. ...
TFIID is an essential basal transcription factor, crucial for RNA polymerase II (pol II) promoter recognition and transcription initiation. The TFIID complex consists of the TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs) that contain intrinsically disordered regions (IDRs) with currently unknown functions. Here, we show that a conserved IDR drives TAF2 condensation in nuclear speckles, independently of other TFIID subunits. Quantitative mass spectrometry analyses reveal that the TAF2 IDR specifically interacts with the nuclear speckle and spliceosome-associated protein SRRM2. Consequently, TAF2 recruits SRRM2 to TFIID to form non-canonical TFIID-SRRM2 complexes. Reduced SRRM2 recruitment elicits alternative splicing events in RNAs coding for proteins involved in transcription and transmembrane transport. Further, genome-wide binding analyses suggest TAF2 shuttling between nuclear speckles and pol II promoters. This study identifies an IDR of the basal transcription machinery as a molecular guide for protein partitioning into nuclear compartments, controlling protein complex composition and pre-mRNA splicing.
... Studies on IgM splicing have highlighted the indispensability of RS domains, as the exonic splicing enhancer presents sub-optimal binding sites for RRMs (44)(45)(46). RS domains are also present in a larger family called SR-like proteins, which are extensively involved in RNA processing (47)(48)(49)(50). These observations hint at the potential role of RS domains as crucial RNA-binding components. ...
... Studies on IgM splicing have highlighted the indispensability of RS domains, as the exonic splicing enhancer presents sub-optimal binding sites for RRMs (44)(45)(46). RS domains are also present in a larger family called SR-like proteins, which are extensively involved in RNA processing (47)(48)(49)(50). These observations hint at the potential role of RS domains as crucial RNA-binding components. ...
SRSF1 governs splicing of over 1,500 mRNA transcripts. SRSF1 contains two RNA-recognition motifs (RRMs) and a C-terminal Arg/Ser-rich region (RS). It has been thought that SRSF1 RRMs exclusively recognize single-stranded exonic splicing enhancers, while RS lacks RNA-binding specificity. With our success in solving the insolubility problem of SRSF1, we can explore the unknown RNA-binding landscape of SRSF1. We find that SRSF1 RS prefers purine over pyrimidine. Moreover, SRSF1 binds to the G-quadruplex (GQ) from the ARPC2 mRNA, with both RRMs and RS being crucial. Our binding assays show that the traditional RNA-binding sites on the RRM tandem and the Arg in RS are responsible for GQ binding. Interestingly, our FRET and circular dichroism data reveal that SRSF1 unfolds the ARPC2 GQ, with RS leading unfolding and RRMs aiding. Our saturation transfer difference NMR results discover that Arg residues in SRSF1 RS interact with the guanine base but other nucleobases, underscoring the uniqueness of the Arg/guanine interaction. Our luciferase assays confirm that SRSF1 can alleviate the inhibitory effect of GQ on gene expression in the cell. Given the prevalence of RNA GQ and SR proteins, our findings unveil unexplored SR protein functions with broad implications in RNA splicing and translation.
... Nuclear speckles are typical membrane-less biomolecular condensates located in the interchromatin-space of the nucleus, serving as the reservoir for RNA processing and splicing factors for mRNA alternative splicing [87]. Nuclear speckle assembly is driven by two large scaffold proteins, SRRM2 and SON [88,89]. Direct evidence that nuclear speckles arise through LLPS is lacking, which is at least partially due to technical challenges. ...
Biomolecular condensates are dynamic non-membrane-bound macromolecular high-order assemblies that participate in a growing list of cellular processes, such as transcription, the cell cycle, etc. Disturbed dynamics of biomolecular condensates are associated with many diseases, including cancer and neurodegeneration. Extensive efforts have been devoted to uncovering the molecular and biochemical grammar governing the dynamics of biomolecular condensates and establishing the critical roles of protein posttranslational modifications (PTMs) in this process. Here, we summarize the regulatory roles of ubiquitination (a major form of cellular PTM) in the dynamics of biomolecular condensates. We propose that these regulatory mechanisms can be harnessed to combat many diseases.
... For cytosolic-nuclear/dimethyl-downregulated sites (Fig. S11F), we identified seven phosphorylation sites in different proteins (S176 on Acin1, S1870 on Akap13, S133 on Eef1d, S1969 on Jmjd1c, S88 on Lrrfip1, S216 on Mllt4, and S2624 on Srrm2). In accordance with a recently published study identifying mRNA processing-related proteins as putative substrates of Dclk1 [81], five of these proteins were shown to be connected to splicing (Acin1 and Srrm2) [83,84], translation (Eef1d) [85], and transcriptional regulation (Lrrfip1 and Jmjdc1) [86,87]. Furthermore, Mllt4/ AFDN was shown to be involved in PDGF signaling [88] and cell division [89], in line with the processes found to be regulated on both the protein and phosphorylation site level in our datasets (Figs. 2, 3). ...
Oligodendrocytes are generated via a two-step mechanism from pluripotent neural stem cells (NSCs): after differentiation of NSCs to oligodendrocyte precursor/NG2 cells (OPCs), they further develop into mature oligodendrocytes. The first step of this differentiation process is only incompletely understood. In this study, we utilized the neurosphere assay to investigate NSC to OPC differentiation in a time course-dependent manner by mass spectrometry-based (phospho-) proteomics. We identify doublecortin-like kinase 1 (Dclk1) as one of the most prominently regulated proteins in both datasets, and show that it undergoes a gradual transition between its short/long isoform during NSC to OPC differentiation. This is regulated by phosphorylation of its SP-rich region, resulting in inhibition of proteolytic Dclk1 long cleavage, and therefore Dclk1 short generation. Through interactome analyses of different Dclk1 isoforms by proximity biotinylation, we characterize their individual putative interaction partners and substrates. All data are available via ProteomeXchange with identifier PXD040652.
... To be able to visualize nuclear speckles in live cells we RFPtagged SRRM2 in the SMNDC1-GFP-tagged cells (Fig. 1g). SRRM2 is the target of the SC35 antibody 45 and scaffolding protein of nuclear speckles 46 . ...
SMNDC1 is a Tudor domain protein that recognizes di-methylated arginines and controls gene expression as an essential splicing factor. Here, we study the specific contributions of the SMNDC1 Tudor domain to protein-protein interactions, subcellular localization, and molecular function. To perturb the protein function in cells, we develop small molecule inhibitors targeting the dimethylarginine binding pocket of the SMNDC1 Tudor domain. We find that SMNDC1 localizes to phase-separated membraneless organelles that partially overlap with nuclear speckles. This condensation behavior is driven by the unstructured C-terminal region of SMNDC1, depends on RNA interaction and can be recapitulated in vitro. Inhibitors of the protein’s Tudor domain drastically alter protein-protein interactions and subcellular localization, causing splicing changes for SMNDC1-dependent genes. These compounds will enable further pharmacological studies on the role of SMNDC1 in the regulation of nuclear condensates, gene regulation and cell identity.
