logo logo
p53 isoforms differentially impact on the POLι dependent DNA damage tolerance pathway. Cell death & disease The recently discovered p53-dependent DNA damage tolerance (DDT) pathway relies on its biochemical activities in DNA-binding, oligomerization, as well as complex formation with the translesion synthesis (TLS) polymerase iota (POLι). These p53-POLι complexes slow down nascent DNA synthesis for safe, homology-directed bypass of DNA replication barriers. In this study, we demonstrate that the alternative p53-isoforms p53β, p53γ, Δ40p53α, Δ133p53α, and Δ160p53α differentially affect this p53-POLι-dependent DDT pathway originally described for canonical p53α. We show that the C-terminal isoforms p53β and p53γ, comprising a truncated oligomerization domain (OD), bind PCNA. Conversely, N-terminally truncated isoforms have a reduced capacity to engage in this interaction. Regardless of the specific loss of biochemical activities required for this DDT pathway, all alternative isoforms were impaired in promoting POLι recruitment to PCNA in the chromatin and in decelerating DNA replication under conditions of enforced replication stress after Mitomycin C (MMC) treatment. Consistent with this, all alternative p53-isoforms no longer stimulated recombination, i.e., bypass of endogenous replication barriers. Different from the other isoforms, Δ133p53α and Δ160p53α caused a severe DNA replication problem, namely fork stalling even in untreated cells. Co-expression of each alternative p53-isoform together with p53α exacerbated the DDT pathway defects, unveiling impaired POLι recruitment and replication deceleration already under unperturbed conditions. Such an inhibitory effect on p53α was particularly pronounced in cells co-expressing Δ133p53α or Δ160p53α. Notably, this effect became evident after the expression of the isoforms in tumor cells, as well as after the knockdown of endogenous isoforms in human hematopoietic stem and progenitor cells. In summary, mimicking the situation found to be associated with many cancer types and stem cells, i.e., co-expression of alternative p53-isoforms with p53α, carved out interference with p53α functions in the p53-POLι-dependent DDT pathway. 10.1038/s41419-021-04224-3
Effect of p53 and its N-terminally truncated isoform, Δ40p53, on breast cancer migration and invasion. Zhang Xiajie,Groen Kira,Morten Brianna C,Steffens Reinhardt Luiza,Campbell Hamish G,Braithwaite Antony W,Bourdon Jean-Christophe,Avery-Kiejda Kelly A Molecular oncology Breast cancer is the most diagnosed malignancy in women, with over half a million women dying from this disease each year. In our previous studies, ∆40p53, an N-terminally truncated p53 isoform, was found to be upregulated in breast cancers, and a high ∆40p53 : p53α ratio was linked with worse disease-free survival. Although p53α inhibits cancer migration and invasion, little is known about the role of ∆40p53 in regulating these metastasis-related processes and its role in contributing to worse prognosis. The aim of this study was to assess the role of ∆40p53 in breast cancer migration and invasion. A relationship between Δ40p53 and gene expression profiles was identified in oestrogen-receptor-positive breast cancer specimens. To further evaluate the role of Δ40p53 in oestrogen-receptor-positive breast cancer, MCF-7 and ZR75-1 cell lines were transduced to knockdown p53α or Δ40p53 and overexpress Δ40p53. Proliferation, migration and invasion were assessed in the transduced sublines, and gene expression was assessed through RNA-sequencing and validated by reverse-transcription quantitative PCR. Knockdown of both p53α and ∆40p53 resulted in increased proliferation, whereas overexpression of ∆40p53 reduced proliferation rates. p53α knockdown was also associated with increased cell mobility. ∆40p53 overexpression reduced both migratory and invasive properties of the transduced cells. Phenotypic findings are supported by gene expression data, including differential expression of LRG1, HYOU1, UBE2QL1, SERPINA5 and PCDH7. Taken together, these results suggest that, at the basal level, ∆40p53 works similarly to p53α in suppressing cellular mobility and proliferation, although the role of Δ40p53 may be cell context-specific. 10.1002/1878-0261.13118
Coordinated alterations in RNA splicing and epigenetic regulation drive leukaemogenesis. Nature Transcription and pre-mRNA splicing are key steps in the control of gene expression and mutations in genes regulating each of these processes are common in leukaemia. Despite the frequent overlap of mutations affecting epigenetic regulation and splicing in leukaemia, how these processes influence one another to promote leukaemogenesis is not understood and, to our knowledge, there is no functional evidence that mutations in RNA splicing factors initiate leukaemia. Here, through analyses of transcriptomes from 982 patients with acute myeloid leukaemia, we identified frequent overlap of mutations in IDH2 and SRSF2 that together promote leukaemogenesis through coordinated effects on the epigenome and RNA splicing. Whereas mutations in either IDH2 or SRSF2 imparted distinct splicing changes, co-expression of mutant IDH2 altered the splicing effects of mutant SRSF2 and resulted in more profound splicing changes than either mutation alone. Consistent with this, co-expression of mutant IDH2 and SRSF2 resulted in lethal myelodysplasia with proliferative features in vivo and enhanced self-renewal in a manner not observed with either mutation alone. IDH2 and SRSF2 double-mutant cells exhibited aberrant splicing and reduced expression of INTS3, a member of the integrator complex, concordant with increased stalling of RNA polymerase II (RNAPII). Aberrant INTS3 splicing contributed to leukaemogenesis in concert with mutant IDH2 and was dependent on mutant SRSF2 binding to cis elements in INTS3 mRNA and increased DNA methylation of INTS3. These data identify a pathogenic crosstalk between altered epigenetic state and splicing in a subset of leukaemias, provide functional evidence that mutations in splicing factors drive myeloid malignancy development, and identify spliceosomal changes as a mediator of IDH2-mutant leukaemogenesis. 10.1038/s41586-019-1618-0
Modulation of splicing catalysis for therapeutic targeting of leukemia with mutations in genes encoding spliceosomal proteins. Lee Stanley Chun-Wei,Dvinge Heidi,Kim Eunhee,Cho Hana,Micol Jean-Baptiste,Chung Young Rock,Durham Benjamin H,Yoshimi Akihide,Kim Young Joon,Thomas Michael,Lobry Camille,Chen Chun-Wei,Pastore Alessandro,Taylor Justin,Wang Xujun,Krivtsov Andrei,Armstrong Scott A,Palacino James,Buonamici Silvia,Smith Peter G,Bradley Robert K,Abdel-Wahab Omar Nature medicine Mutations in genes encoding splicing factors (which we refer to as spliceosomal genes) are commonly found in patients with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). These mutations recurrently affect specific amino acid residues, leading to perturbed normal splice site and exon recognition. Spliceosomal gene mutations are always heterozygous and rarely occur together with one another, suggesting that cells may tolerate only a partial deviation from normal splicing activity. To test this hypothesis, we engineered mice to express a mutated allele of serine/arginine-rich splicing factor 2 (Srsf2(P95H))-which commonly occurs in individuals with MDS and AML-in an inducible, hemizygous manner in hematopoietic cells. These mice rapidly succumbed to fatal bone marrow failure, demonstrating that Srsf2-mutated cells depend on the wild-type Srsf2 allele for survival. In the context of leukemia, treatment with the spliceosome inhibitor E7107 (refs. 7,8) resulted in substantial reductions in leukemic burden, specifically in isogenic mouse leukemias and patient-derived xenograft AMLs carrying spliceosomal mutations. Whereas E7107 treatment of mice resulted in widespread intron retention and cassette exon skipping in leukemic cells regardless of Srsf2 genotype, the magnitude of splicing inhibition following E7107 treatment was greater in Srsf2-mutated than in Srsf2-wild-type leukemia, consistent with the differential effect of E7107 on survival. Collectively, these data provide genetic and pharmacologic evidence that leukemias with spliceosomal gene mutations are preferentially susceptible to additional splicing perturbations in vivo as compared to leukemias without such mutations. Modulation of spliceosome function may thus provide a new therapeutic avenue in genetically defined subsets of individuals with MDS or AML. 10.1038/nm.4097
SRSF1 promotes vascular smooth muscle cell proliferation through a Δ133p53/EGR1/KLF5 pathway. Xie Ning,Chen Min,Dai Rilei,Zhang Yan,Zhao Hanqing,Song Zhiming,Zhang Lufeng,Li Zhenyan,Feng Yuanqing,Gao Hua,Wang Li,Zhang Ting,Xiao Rui-Ping,Wu Jianxin,Cao Chun-Mei Nature communications Though vascular smooth muscle cell (VSMC) proliferation underlies all cardiovascular hyperplastic disorders, our understanding of the molecular mechanisms responsible for this cellular process is still incomplete. Here we report that SRSF1 (serine/arginine-rich splicing factor 1), an essential splicing factor, promotes VSMC proliferation and injury-induced neointima formation. Vascular injury in vivo and proliferative stimuli in vitro stimulate SRSF1 expression. Mice lacking SRSF1 specifically in SMCs develop less intimal thickening after wire injury. Expression of SRSF1 in rat arteries enhances neointima formation. SRSF1 overexpression increases, while SRSF1 knockdown suppresses the proliferation and migration of cultured human aortic and coronary arterial SMCs. Mechanistically, SRSF1 favours the induction of a truncated p53 isoform, Δ133p53, which has an equal proliferative effect and in turn transcriptionally activates Krüppel-like factor 5 (KLF5) via the Δ133p53-EGR1 complex, resulting in an accelerated cell-cycle progression and increased VSMC proliferation. Our study provides a potential therapeutic target for vascular hyperplastic disease. 10.1038/ncomms16016
SRSF1-dependent nuclear export inhibition of C9ORF72 repeat transcripts prevents neurodegeneration and associated motor deficits. Nature communications Hexanucleotide repeat expansions in the C9ORF72 gene are the commonest known genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Expression of repeat transcripts and dipeptide repeat proteins trigger multiple mechanisms of neurotoxicity. How repeat transcripts get exported from the nucleus is unknown. Here, we show that depletion of the nuclear export adaptor SRSF1 prevents neurodegeneration and locomotor deficits in a Drosophila model of C9ORF72-related disease. This intervention suppresses cell death of patient-derived motor neuron and astrocytic-mediated neurotoxicity in co-culture assays. We further demonstrate that either depleting SRSF1 or preventing its interaction with NXF1 specifically inhibits the nuclear export of pathological C9ORF72 transcripts, the production of dipeptide-repeat proteins and alleviates neurotoxicity in Drosophila, patient-derived neurons and neuronal cell models. Taken together, we show that repeat RNA-sequestration of SRSF1 triggers the NXF1-dependent nuclear export of C9ORF72 transcripts retaining expanded hexanucleotide repeats and reveal a novel promising therapeutic target for neuroprotection. 10.1038/ncomms16063
Genomics of chronic neutrophilic leukemia. Maxson Julia E,Tyner Jeffrey W Blood Chronic neutrophilic leukemia (CNL) is a distinct myeloproliferative neoplasm with a high prevalence (>80%) of mutations in the colony-stimulating factor 3 receptor (). These mutations activate the receptor, leading to the proliferation of neutrophils that are a hallmark of CNL. Recently, the World Health Organization guidelines have been updated to include mutations as part of the diagnostic criteria for CNL. Because of the high prevalence of mutations in CNL, it is tempting to think of this disease as being solely driven by this genetic lesion. However, recent additional genomic characterization demonstrates that CNL has much in common with other chronic myeloid malignancies at the genetic level, such as the clinically related diagnosis atypical chronic myeloid leukemia. These commonalities include mutations in , spliceosome proteins (, ), and epigenetic modifiers (, ). Some of these same mutations also have been characterized as frequent events in clonal hematopoiesis of indeterminate potential, suggesting a more complex disease evolution than was previously understood and raising the possibility that an age-related clonal process of preleukemic cells could precede the development of CNL. The order of acquisition of mutations relative to mutations in , epigenetic modifiers, or the spliceosome has been determined only in isolated case reports; thus, further work is needed to understand the impact of mutation chronology on the clonal evolution and progression of CNL. Understanding the complete landscape and chronology of genomic events in CNL will help in the development of improved therapeutic strategies for this patient population. 10.1182/blood-2016-10-695981
NEDDylation promotes stress granule assembly. Jayabalan Aravinth Kumar,Sanchez Anthony,Park Ra Young,Yoon Sang Pil,Kang Gum-Yong,Baek Je-Hyun,Anderson Paul,Kee Younghoon,Ohn Takbum Nature communications Stress granules (SGs) harbour translationally stalled messenger ribonucleoproteins and play important roles in regulating gene expression and cell fate. Here we show that neddylation promotes SG assembly in response to arsenite-induced oxidative stress. Inhibition or depletion of key components of the neddylation machinery concomitantly inhibits stress-induced polysome disassembly and SG assembly. Affinity purification and subsequent mass-spectrometric analysis of Nedd8-conjugated proteins from translationally stalled ribosomal fractions identified ribosomal proteins, translation factors and RNA-binding proteins (RBPs), including SRSF3, a previously known SG regulator. We show that SRSF3 is selectively neddylated at Lys85 in response to arsenite. A non-neddylatable SRSF3 (K85R) mutant do not prevent arsenite-induced polysome disassembly, but fails to support the SG assembly, suggesting that the neddylation pathway plays an important role in SG assembly. 10.1038/ncomms12125
Loss of 5-methylcytosine alters the biogenesis of vault-derived small RNAs to coordinate epidermal differentiation. Nature communications The presence and absence of RNA modifications regulates RNA metabolism by modulating the binding of writer, reader, and eraser proteins. For 5-methylcytosine (mC) however, it is largely unknown how it recruits or repels RNA-binding proteins. Here, we decipher the consequences of mC deposition into the abundant non-coding vault RNA VTRNA1.1. Methylation of cytosine 69 in VTRNA1.1 occurs frequently in human cells, is exclusively mediated by NSUN2, and determines the processing of VTRNA1.1 into small-vault RNAs (svRNAs). We identify the serine/arginine rich splicing factor 2 (SRSF2) as a novel VTRNA1.1-binding protein that counteracts VTRNA1.1 processing by binding the non-methylated form with higher affinity. Both NSUN2 and SRSF2 orchestrate the production of distinct svRNAs. Finally, we discover a functional role of svRNAs in regulating the epidermal differentiation programme. Thus, our data reveal a direct role for mC in the processing of VTRNA1.1 that involves SRSF2 and is crucial for efficient cellular differentiation. 10.1038/s41467-019-10020-7
Two splice-factor mutant leukemia subgroups uncovered at the boundaries of MDS and AML using combined gene expression and DNA-methylation profiling. Taskesen Erdogan,Havermans Marije,van Lom Kirsten,Sanders Mathijs A,van Norden Yvette,Bindels Eric,Hoogenboezem Remco,Reinders Marcel J T,Figueroa Maria E,Valk Peter J M,Löwenberg Bob,Melnick Ari,Delwel Ruud Blood Mutations in splice factor (SF) genes occur more frequently in myelodysplastic syndromes (MDS) than in acute myeloid leukemias (AML). We sequenced complementary DNA from bone marrow of 47 refractory anemia with excess blasts (RAEB) patients, 29 AML cases with low marrow blast cell count, and 325 other AML patients and determined the presence of SF-hotspot mutations in SF3B1, U2AF35, and SRSF2. SF mutations were found in 10 RAEB, 12 AML cases with low marrow blast cell count, and 25 other AML cases. Our study provides evidence that SF-mutant RAEB and SF-mutant AML are clinically, cytologically, and molecularly highly similar. An integrated analysis of genomewide messenger RNA (mRNA) expression profiling and DNA-methylation profiling data revealed 2 unique patient clusters highly enriched for SF-mutant RAEB/AML. The combined genomewide mRNA expression profiling/DNA-methylation profiling signatures revealed 1 SF-mutant patient cluster with an erythroid signature. The other SF-mutant patient cluster was enriched for NRAS/KRAS mutations and showed an inferior survival. We conclude that SF-mutant RAEB/AML constitutes a related disorder overriding the artificial separation between AML and MDS, and that SF-mutant RAEB/AML is composed of 2 molecularly and clinically distinct subgroups. We conclude that SF-mutant disorders should be considered as myeloid malignancies that transcend the boundaries of AML and MDS. 10.1182/blood-2013-07-512855
Antisense oligonucleotide-mediated MDM4 exon 6 skipping impairs tumor growth. Dewaele Michael,Tabaglio Tommaso,Willekens Karen,Bezzi Marco,Teo Shun Xie,Low Diana H P,Koh Cheryl M,Rambow Florian,Fiers Mark,Rogiers Aljosja,Radaelli Enrico,Al-Haddawi Muthafar,Tan Soo Yong,Hermans Els,Amant Frederic,Yan Hualong,Lakshmanan Manikandan,Koumar Ratnacaram Chandrahas,Lim Soon Thye,Derheimer Frederick A,Campbell Robert M,Bonday Zahid,Tergaonkar Vinay,Shackleton Mark,Blattner Christine,Marine Jean-Christophe,Guccione Ernesto The Journal of clinical investigation MDM4 is a promising target for cancer therapy, as it is undetectable in most normal adult tissues but often upregulated in cancer cells to dampen p53 tumor-suppressor function. The mechanisms that underlie MDM4 upregulation in cancer cells are largely unknown. Here, we have shown that this key oncogenic event mainly depends on a specific alternative splicing switch. We determined that while a nonsense-mediated, decay-targeted isoform of MDM4 (MDM4-S) is produced in normal adult tissues as a result of exon 6 skipping, enhanced exon 6 inclusion leads to expression of full-length MDM4 in a large number of human cancers. Although this alternative splicing event is likely regulated by multiple splicing factors, we identified the SRSF3 oncoprotein as a key enhancer of exon 6 inclusion. In multiple human melanoma cell lines and in melanoma patient-derived xenograft (PDX) mouse models, antisense oligonucleotide-mediated (ASO-mediated) skipping of exon 6 decreased MDM4 abundance, inhibited melanoma growth, and enhanced sensitivity to MAPK-targeting therapeutics. Additionally, ASO-based MDM4 targeting reduced diffuse large B cell lymphoma PDX growth. As full-length MDM4 is enhanced in multiple human tumors, our data indicate that this strategy is applicable to a wide range of tumor types. We conclude that enhanced MDM4 exon 6 inclusion is a common oncogenic event and has potential as a clinically compatible therapeutic target. 10.1172/JCI82534
Poly-dipeptides encoded by the C9orf72 repeats bind nucleoli, impede RNA biogenesis, and kill cells. Kwon Ilmin,Xiang Siheng,Kato Masato,Wu Leeju,Theodoropoulos Pano,Wang Tao,Kim Jiwoong,Yun Jonghyun,Xie Yang,McKnight Steven L Science (New York, N.Y.) Many RNA regulatory proteins controlling pre-messenger RNA splicing contain serine:arginine (SR) repeats. Here, we found that these SR domains bound hydrogel droplets composed of fibrous polymers of the low-complexity domain of heterogeneous ribonucleoprotein A2 (hnRNPA2). Hydrogel binding was reversed upon phosphorylation of the SR domain by CDC2-like kinases 1 and 2 (CLK1/2). Mutated variants of the SR domains changing serine to glycine (SR-to-GR variants) also bound to hnRNPA2 hydrogels but were not affected by CLK1/2. When expressed in mammalian cells, these variants bound nucleoli. The translation products of the sense and antisense transcripts of the expansion repeats associated with the C9orf72 gene altered in neurodegenerative disease encode GRn and PRn repeat polypeptides. Both peptides bound to hnRNPA2 hydrogels independent of CLK1/2 activity. When applied to cultured cells, both peptides entered cells, migrated to the nucleus, bound nucleoli, and poisoned RNA biogenesis, which caused cell death. 10.1126/science.1254917
Genetic variation and RNA structure regulate microRNA biogenesis. Nature communications MiRNA biogenesis is highly regulated at the post-transcriptional level; however, the role of sequence and secondary RNA structure in this process has not been extensively studied. A single G to A substitution present in the terminal loop of pri-mir-30c-1 in breast and gastric cancer patients had been previously described to result in increased levels of mature miRNA. Here, we report that this genetic variant directly affects Drosha-mediated processing of pri-mir-30c-1 in vitro and in cultured cells. Structural analysis of this variant revealed an altered RNA structure that facilitates the interaction with SRSF3, an SR protein family member that promotes pri-miRNA processing. Our results are compatible with a model whereby a genetic variant in pri-mir-30c-1 leads to a secondary RNA structure rearrangement that facilitates binding of SRSF3 resulting in increased levels of miR-30c. These data highlight that primary sequence determinants and RNA structure are key regulators of miRNA biogenesis. 10.1038/ncomms15114
Interactome analysis brings splicing into focus. Dominguez Daniel,Burge Christopher B Genome biology The spliceosome is a huge molecular machine that assembles dynamically onto its pre-mRNA substrates. A new study based on interactome analysis provides clues about how splicing-regulatory proteins modulate assembly of the spliceosome to either activate or repress splicing.Please see related Research article: http://www.genomebiology.com/2015/16/1/119/abstract. 10.1186/s13059-015-0707-0
Quality control of spliced mRNAs requires the shuttling SR proteins Gbp2 and Hrb1. Hackmann Alexandra,Wu Haijia,Schneider Ulla-Maria,Meyer Katja,Jung Klaus,Krebber Heike Nature communications Eukaryotic cells have to prevent the export of unspliced pre-mRNAs until intron removal is completed to avoid the expression of aberrant and potentially harmful proteins. Only mature mRNAs associate with the export receptor Mex67/TAP and enter the cytoplasm. Here we show that the two shuttling serine/arginine (SR)-proteins Gbp2 and Hrb1 are key surveillance factors for the selective export of spliced mRNAs in yeast. Their absence leads to the significant leakage of unspliced pre-mRNAs into the cytoplasm. They bind to pre-mRNAs and the spliceosome during splicing, where they are necessary for the surveillance of splicing and the stable binding of the TRAMP complex to spliceosome-bound transcripts. Faulty transcripts are marked for their degradation at the nuclear exosome. On correct mRNAs the SR proteins recruit Mex67 upon completion of splicing to allow a quality controlled nuclear export. Altogether, these data identify a role for shuttling SR proteins in mRNA surveillance and nuclear mRNA quality control. 10.1038/ncomms4123
Sequestration of multiple RNA recognition motif-containing proteins by C9orf72 repeat expansions. Cooper-Knock Johnathan,Walsh Matthew J,Higginbottom Adrian,Robin Highley J,Dickman Mark J,Edbauer Dieter,Ince Paul G,Wharton Stephen B,Wilson Stuart A,Kirby Janine,Hautbergue Guillaume M,Shaw Pamela J Brain : a journal of neurology GGGGCC repeat expansions of C9orf72 represent the most common genetic variant of amyotrophic lateral sclerosis and frontotemporal degeneration, but the mechanism of pathogenesis is unclear. Recent reports have suggested that the transcribed repeat might form toxic RNA foci that sequester various RNA processing proteins. Consensus as to the identity of the binding partners is missing and whole neuronal proteome investigation is needed. Using RNA fluorescence in situ hybridization we first identified nuclear and cytoplasmic RNA foci in peripheral and central nervous system biosamples from patients with amyotrophic lateral sclerosis with a repeat expansion of C9orf72 (C9orf72+), but not from those patients without a repeat expansion of C9orf72 (C9orf72-) or control subjects. Moreover, in the cases examined, the distribution of foci-positive neurons correlated with the clinical phenotype (t-test P < 0.05). As expected, RNA foci are ablated by RNase treatment. Interestingly, we identified foci in fibroblasts from an asymptomatic C9orf72+ carrier. We next performed pulldown assays, with GGGGCC5, in conjunction with mass spectrometry analysis, to identify candidate binding partners of the GGGGCC repeat expansion. Proteins containing RNA recognition motifs and involved in splicing, messenger RNA nuclear export and/or translation were significantly enriched. Immunohistochemistry in central nervous system tissue from C9orf72+ patients with amyotrophic lateral sclerosis demonstrated co-localization of RNA foci with SRSF2, hnRNP H1/F, ALYREF and hnRNP A1 in cerebellar granule cells and with SRSF2, hnRNP H1/F and ALYREF in motor neurons, the primary target of pathology in amyotrophic lateral sclerosis. Direct binding of proteins to GGGGCC repeat RNA was confirmed in vitro by ultraviolet-crosslinking assays. Co-localization was only detected in a small proportion of RNA foci, suggesting dynamic sequestration rather than irreversible binding. Additional immunohistochemistry demonstrated that neurons with and without RNA foci were equally likely to show nuclear depletion of TDP-43 (χ(2) P = 0.75) or poly-GA dipeptide repeat protein inclusions (χ(2) P = 0.46). Our findings suggest two non-exclusive pathogenic mechanisms: (i) functional depletion of RNA-processing proteins resulting in disruption of messenger RNA splicing; and (ii) licensing of expanded C9orf72 pre-messenger RNA for nuclear export by inappropriate association with messenger RNA export adaptor protein(s) leading to cytoplasmic repeat associated non-ATG translation and formation of potentially toxic dipeptide repeat protein. 10.1093/brain/awu120
Mutually exclusive acetylation and ubiquitylation of the splicing factor SRSF5 control tumor growth. Chen Yuhan,Huang Qingyang,Liu Wen,Zhu Qiong,Cui Chun-Ping,Xu Liang,Guo Xing,Wang Ping,Liu Jingwen,Dong Guanglong,Wei Wenyi,Liu Cui Hua,Feng Zhichun,He Fuchu,Zhang Lingqiang Nature communications Most tumor cells take up more glucose than normal cells. Splicing dysregulation is one of the molecular hallmarks of cancer. However, the role of splicing factor in glucose metabolism and tumor development remains poorly defined. Here, we show that upon glucose intake, the splicing factor SRSF5 is specifically induced through Tip60-mediated acetylation on K125, which antagonizes Smurf1-mediated ubiquitylation. SRSF5 promotes the alternative splicing of CCAR1 to produce CCAR1S proteins, which promote tumor growth by enhancing glucose consumption and acetyl-CoA production. Conversely, upon glucose starvation, SRSF5 is deacetylated by HDAC1, and ubiquitylated by Smurf1 on the same lysine, resulting in proteasomal degradation of SRSF5. The CCAR1L proteins accumulate to promote apoptosis. Importantly, SRSF5 is hyperacetylated and upregulated in human lung cancers, which correlates with increased CCAR1S expression and tumor progression. Thus, SRSF5 responds to high glucose to promote cancer development, and SRSF5-CCAR1 axis may be valuable targets for cancer therapeutics. 10.1038/s41467-018-04815-3
RECQ5-dependent SUMOylation of DNA topoisomerase I prevents transcription-associated genome instability. Li Min,Pokharel Subhash,Wang Jiin-Tarng,Xu Xiaohua,Liu Yilun Nature communications DNA topoisomerase I (TOP1) has an important role in maintaining DNA topology by relaxing supercoiled DNA. Here we show that the K391 and K436 residues of TOP1 are SUMOylated by the PIAS1-SRSF1 E3 ligase complex in the chromatin fraction containing active RNA polymerase II (RNAPIIo). This modification is necessary for the binding of TOP1 to RNAPIIo and for the recruitment of RNA splicing factors to the actively transcribed chromatin, thereby reducing the formation of R-loops that lead to genome instability. RECQ5 helicase promotes TOP1 SUMOylation by facilitating the interaction between PIAS1, SRSF1 and TOP1. Unexpectedly, the topoisomerase activity is compromised by K391/K436 SUMOylation, and this provides the first in vivo evidence that TOP1 activity is negatively regulated at transcriptionally active chromatin to prevent TOP1-induced DNA damage. Therefore, our data provide mechanistic insight into how TOP1 SUMOylation contributes to genome maintenance during transcription. 10.1038/ncomms7720
Molecular Mechanisms for CFIm-Mediated Regulation of mRNA Alternative Polyadenylation. Zhu Yong,Wang Xiuye,Forouzmand Elmira,Jeong Joshua,Qiao Feng,Sowd Gregory A,Engelman Alan N,Xie Xiaohui,Hertel Klemens J,Shi Yongsheng Molecular cell Alternative mRNA processing is a critical mechanism for proteome expansion and gene regulation in higher eukaryotes. The SR family proteins play important roles in splicing regulation. Intriguingly, mammalian genomes encode many poorly characterized SR-like proteins, including subunits of the mRNA 3'-processing factor CFIm, CFIm68 and CFIm59. Here we demonstrate that CFIm functions as an enhancer-dependent activator of mRNA 3' processing. CFIm regulates global alternative polyadenylation (APA) by specifically binding and activating enhancer-containing poly(A) sites (PASs). Importantly, the CFIm activator functions are mediated by the arginine-serine repeat (RS) domains of CFIm68/59, which bind specifically to an RS-like region in the CPSF subunit Fip1, and this interaction is inhibited by CFIm68/59 hyper-phosphorylation. The remarkable functional similarities between CFIm and SR proteins suggest that interactions between RS-like domains in regulatory and core factors may provide a common activation mechanism for mRNA 3' processing, splicing, and potentially other steps in RNA metabolism. 10.1016/j.molcel.2017.11.031
A Coiled-Coil Domain Containing 50 Splice Variant Is Modulated by Serine/Arginine-Rich Splicing Factor 3 and Promotes Hepatocellular Carcinoma in Mice by the Ras Signaling Pathway. Wang Hong,Zhang Chris Zhiyi,Lu Shi-Xun,Zhang Mei-Fang,Liu Li-Li,Luo Rong-Zhen,Yang Xia,Wang Chun-Hua,Chen Shi-Lu,He Yang-Fan,Xie Dan,Xu Rui-Hua,Yun Jing-Ping Hepatology (Baltimore, Md.) Deregulation of alternative splicing contributes to the malignant progression of cancer. Little is known about the significant alternative splicing events in hepatocellular carcinoma (HCC). High-throughput sequencing revealed that coiled-coil domain containing 50 (CCDC50) pre-mRNA is aberrantly spliced in 50% of our HCC cases. A BaseScope assay was performed to examine the expression of CCDC50S (a truncated oncogenic splice variant) in HCC tissues. Compared with benign liver tumors and several other types of solid tumors, CCDC50S mRNA was up-regulated in HCC, with a diagnostic potential (sensitivity, 0.711; specificity, 0.793). High expression of CCDC50S mRNA in HCC was significantly correlated with poor tumor differentiation, advanced tumor node metastasis (TNM) stage, and unfavorable prognosis. Overexpression of CCDC50S exerted tumorigenic activities that promoted HCC growth and metastasis by activation of Ras/forkhead box protein O4 (Foxo4) signaling. Either suppression of mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) phosphorylation or overexpression of Foxo4 markedly attenuated CCDC50S-mediated phenotypes. Furthermore, serine- and arginine-rich splicing factor 3 (SRSF3) directly bound to CCDC50S mRNA to maintain its stability in the cytoplasm. The cytosolic retention of SRSF3 was mediated by the interaction of hepatitis B virus-encoded X protein (HBx) and 14-3-3β. Ectopic HBx expression induced expression of cytosolic SRSF3 and CCDC50S. Conclusion: Our study provided compelling evidence that up-regulation of CCDC50S was modulated by HBx/SRSF3/14-3-3β complex and enhanced oncogenic progression of HCC through the Ras/Foxo4 signaling pathway. These data suggest that CCDC50S may serve as a diagnostic and prognostic biomarker and probably a promising therapeutic target in HCC. 10.1002/hep.30147
Loss of SRSF3 in Cardiomyocytes Leads to Decapping of Contraction-Related mRNAs and Severe Systolic Dysfunction. Ortiz-Sánchez Paula,Villalba-Orero María,López-Olañeta Marina M,Larrasa-Alonso Javier,Sánchez-Cabo Fátima,Martí-Gómez Carlos,Camafeita Emilio,Gómez-Salinero Jesús M,Ramos-Hernández Laura,Nielsen Peter J,Vázquez Jesús,Müller-McNicoll Michaela,García-Pavía Pablo,Lara-Pezzi Enrique Circulation research RATIONALE:RBPs (RNA binding proteins) play critical roles in the cell by regulating mRNA transport, splicing, editing, and stability. The RBP SRSF3 (serine/arginine-rich splicing factor 3) is essential for blastocyst formation and for proper liver development and function. However, its role in the heart has not been explored. OBJECTIVE:To investigate the role of SRSF3 in cardiac function. METHODS AND RESULTS:Cardiac SRSF3 expression was high at mid gestation and decreased during late embryonic development. Mice lacking SRSF3 in the embryonic heart showed impaired cardiomyocyte proliferation and died in utero. In the adult heart, SRSF3 expression was reduced after myocardial infarction, suggesting a possible role in cardiac homeostasis. To determine the role of this RBP in the adult heart, we used an inducible, cardiomyocyte-specific SRSF3 knockout mouse model. After SRSF3 depletion in cardiomyocytes, mice developed severe systolic dysfunction that resulted in death within 8 days. RNA-Seq analysis revealed downregulation of mRNAs encoding sarcomeric and calcium handling proteins. Cardiomyocyte-specific SRSF3 knockout mice also showed evidence of alternative splicing of mTOR (mammalian target of rapamycin) mRNA, generating a shorter protein isoform lacking catalytic activity. This was associated with decreased phosphorylation of 4E-BP1 (eIF4E-binding protein 1), a protein that binds to eIF4E (eukaryotic translation initiation factor 4E) and prevents mRNA decapping. Consequently, we found increased decapping of mRNAs encoding proteins involved in cardiac contraction. Decapping was partially reversed by mTOR activation. CONCLUSIONS:We show that cardiomyocyte-specific loss of SRSF3 expression results in decapping of critical mRNAs involved in cardiac contraction. The molecular mechanism underlying this effect likely involves the generation of a short mTOR isoform by alternative splicing, resulting in reduced 4E-BP1 phosphorylation. The identification of mRNA decapping as a mechanism of systolic heart failure may open the way to the development of urgently needed therapeutic tools. 10.1161/CIRCRESAHA.118.314515
Telomere dysfunction drives aberrant hematopoietic differentiation and myelodysplastic syndrome. Colla Simona,Ong Derrick Sek Tong,Ogoti Yamini,Marchesini Matteo,Mistry Nipun A,Clise-Dwyer Karen,Ang Sonny A,Storti Paola,Viale Andrea,Giuliani Nicola,Ruisaard Kathryn,Ganan Gomez Irene,Bristow Christopher A,Estecio Marcos,Weksberg David C,Ho Yan Wing,Hu Baoli,Genovese Giannicola,Pettazzoni Piergiorgio,Multani Asha S,Jiang Shan,Hua Sujun,Ryan Michael C,Carugo Alessandro,Nezi Luigi,Wei Yue,Yang Hui,D'Anca Marianna,Zhang Li,Gaddis Sarah,Gong Ting,Horner James W,Heffernan Timothy P,Jones Philip,Cooper Laurence J N,Liang Han,Kantarjian Hagop,Wang Y Alan,Chin Lynda,Bueso-Ramos Carlos,Garcia-Manero Guillermo,DePinho Ronald A Cancer cell Myelodysplastic syndrome (MDS) risk correlates with advancing age, therapy-induced DNA damage, and/or shorter telomeres, but whether telomere erosion directly induces MDS is unknown. Here, we provide the genetic evidence that telomere dysfunction-induced DNA damage drives classical MDS phenotypes and alters common myeloid progenitor (CMP) differentiation by repressing the expression of mRNA splicing/processing genes, including SRSF2. RNA-seq analyses of telomere dysfunctional CMP identified aberrantly spliced transcripts linked to pathways relevant to MDS pathogenesis such as genome stability, DNA repair, chromatin remodeling, and histone modification, which are also enriched in mouse CMP haploinsufficient for SRSF2 and in CD34(+) CMML patient cells harboring SRSF2 mutation. Together, our studies establish an intimate link across telomere biology, aberrant RNA splicing, and myeloid progenitor differentiation. 10.1016/j.ccell.2015.04.007
initiates myeloid bias and myelodysplastic/myeloproliferative syndrome from hemopoietic stem cells. Smeets Monique F,Tan Shuh Ying,Xu Jane Jialu,Anande Govardhan,Unnikrishnan Ashwin,Chalk Alistair M,Taylor Scott R,Pimanda John E,Wall Meaghan,Purton Louise E,Walkley Carl R Blood Mutations in occur in myelodysplastic syndromes (MDS) and MDS/myeloproliferative neoplasms (MPN). mutations cluster at proline 95, with the most frequent mutation being a histidine (P95H) substitution. They undergo positive selection, arise early in the course of disease, and have been identified in age-related clonal hemopoiesis. It is not clear how mutation of modifies hemopoiesis or contributes to the development of myeloid bias or MDS/MPN. Two prior mouse models of mutation have been reported; however, these models do not recapitulate many of the clinical features of -mutant disease and relied on bone marrow (BM) transplantation stress to elicit the reported phenotypes. We describe a new conditional murine mutation model, where the P95H mutation is expressed physiologically and heterozygously from its endogenous locus after Cre activation. Using multiple Cre lines, we demonstrate that during native hemopoiesis (ie, no BM transplantation), the mutation needs to occur within the hemopoietic stem-cell-containing populations to promote myelomonocytic bias and expansion with corresponding transcriptional and RNA splicing changes. With age, nontransplanted animals developed a progressive, transplantable disease characterized by myeloid bias, morphological dysplasia, and monocytosis, hallmarks of MDS/MPN in humans. Analysis of cooccurring mutations within the BM demonstrated the acquisition of additional mutations that are recurrent in humans with mutations. The tractable knock-in model we have generated is highly relevant to human disease and will serve to elucidate the effect of mutations on initiation and maintenance of MDS/MPN. 10.1182/blood-2018-04-845602
SRSF2 Mutations Contribute to Myelodysplasia by Mutant-Specific Effects on Exon Recognition. Kim Eunhee,Ilagan Janine O,Liang Yang,Daubner Gerrit M,Lee Stanley C-W,Ramakrishnan Aravind,Li Yue,Chung Young Rock,Micol Jean-Baptiste,Murphy Michele E,Cho Hana,Kim Min-Kyung,Zebari Ahmad S,Aumann Shlomzion,Park Christopher Y,Buonamici Silvia,Smith Peter G,Deeg H Joachim,Lobry Camille,Aifantis Iannis,Modis Yorgo,Allain Frederic H-T,Halene Stephanie,Bradley Robert K,Abdel-Wahab Omar Cancer cell Mutations affecting spliceosomal proteins are the most common mutations in patients with myelodysplastic syndromes (MDS), but their role in MDS pathogenesis has not been delineated. Here we report that mutations affecting the splicing factor SRSF2 directly impair hematopoietic differentiation in vivo, which is not due to SRSF2 loss of function. By contrast, SRSF2 mutations alter SRSF2's normal sequence-specific RNA binding activity, thereby altering the recognition of specific exonic splicing enhancer motifs to drive recurrent mis-splicing of key hematopoietic regulators. This includes SRSF2 mutation-dependent splicing of EZH2, which triggers nonsense-mediated decay, which, in turn, results in impaired hematopoietic differentiation. These data provide a mechanistic link between a mutant spliceosomal protein, alterations in the splicing of key regulators, and impaired hematopoiesis. 10.1016/j.ccell.2015.04.006
CLK-dependent exon recognition and conjoined gene formation revealed with a novel small molecule inhibitor. Funnell Tyler,Tasaki Shinya,Oloumi Arusha,Araki Shinsuke,Kong Esther,Yap Damian,Nakayama Yusuke,Hughes Christopher S,Cheng S-W Grace,Tozaki Hirokazu,Iwatani Misa,Sasaki Satoshi,Ohashi Tomohiro,Miyazaki Tohru,Morishita Nao,Morishita Daisuke,Ogasawara-Shimizu Mari,Ohori Momoko,Nakao Shoichi,Karashima Masatoshi,Sano Masaya,Murai Aiko,Nomura Toshiyuki,Uchiyama Noriko,Kawamoto Tomohiro,Hara Ryujiro,Nakanishi Osamu,Shumansky Karey,Rosner Jamie,Wan Adrian,McKinney Steven,Morin Gregg B,Nakanishi Atsushi,Shah Sohrab,Toyoshiba Hiroyoshi,Aparicio Samuel Nature communications CDC-like kinase phosphorylation of serine/arginine-rich proteins is central to RNA splicing reactions. Yet, the genomic network of CDC-like kinase-dependent RNA processing events remains poorly defined. Here, we explore the connectivity of genomic CDC-like kinase splicing functions by applying graduated, short-exposure, pharmacological CDC-like kinase inhibition using a novel small molecule (T3) with very high potency, selectivity, and cell-based stability. Using RNA-Seq, we define CDC-like kinase-responsive alternative splicing events, the large majority of which monotonically increase or decrease with increasing CDC-like kinase inhibition. We show that distinct RNA-binding motifs are associated with T3 response in skipped exons. Unexpectedly, we observe dose-dependent conjoined gene transcription, which is associated with motif enrichment in the last and second exons of upstream and downstream partners, respectively. siRNA knockdown of CLK2-associated genes significantly increases conjoined gene formation. Collectively, our results reveal an unexpected role for CDC-like kinase in conjoined gene formation, via regulation of 3'-end processing and associated splicing factors.The phosphorylation of serine/arginine-rich proteins by CDC-like kinase is a central regulatory mechanism for RNA splicing reactions. Here, the authors synthesize a novel small molecule CLK inhibitor and map CLK-responsive alternative splicing events and discover an effect on conjoined gene transcription. 10.1038/s41467-016-0008-7
Modeling RNA-Binding Protein Specificity In Vivo by Precisely Registering Protein-RNA Crosslink Sites. Molecular cell RNA-binding proteins (RBPs) regulate post-transcriptional gene expression by recognizing short and degenerate sequence motifs in their target transcripts, but precisely defining their binding specificity remains challenging. Crosslinking and immunoprecipitation (CLIP) allows for mapping of the exact protein-RNA crosslink sites, which frequently reside at specific positions in RBP motifs at single-nucleotide resolution. Here, we have developed a computational method, named mCross, to jointly model RBP binding specificity while precisely registering the crosslinking position in motif sites. We applied mCross to 112 RBPs using ENCODE eCLIP data and validated the reliability of the discovered motifs by genome-wide analysis of allelic binding sites. Our analyses revealed that the prototypical SR protein SRSF1 recognizes clusters of GGA half-sites in addition to its canonical GGAGGA motif. Therefore, SRSF1 regulates splicing of a much larger repertoire of transcripts than previously appreciated, including HNRNPD and HNRNPDL, which are involved in multivalent protein assemblies and phase separation. 10.1016/j.molcel.2019.02.002
Synthetic Lethal and Convergent Biological Effects of Cancer-Associated Spliceosomal Gene Mutations. Cancer cell Mutations affecting RNA splicing factors are the most common genetic alterations in myelodysplastic syndrome (MDS) patients and occur in a mutually exclusive manner. The basis for the mutual exclusivity of these mutations and how they contribute to MDS is not well understood. Here we report that although different spliceosome gene mutations impart distinct effects on splicing, they are negatively selected for when co-expressed due to aberrant splicing and downregulation of regulators of hematopoietic stem cell survival and quiescence. In addition to this synthetic lethal interaction, mutations in the splicing factors SF3B1 and SRSF2 share convergent effects on aberrant splicing of mRNAs that promote nuclear factor κB signaling. These data identify shared consequences of splicing-factor mutations and the basis for their mutual exclusivity. 10.1016/j.ccell.2018.07.003
Splicing factor SRSF1 controls T cell hyperactivity and systemic autoimmunity. Katsuyama Takayuki,Li Hao,Comte Denis,Tsokos George C,Moulton Vaishali R The Journal of clinical investigation Systemic lupus erythematosus (SLE) is a devastating autoimmune disease in which hyperactive T cells play a critical role. Understanding molecular mechanisms underlying the T cell hyperactivity will lead to identification of specific therapeutic targets. Serine/arginine-rich splicing factor 1 (SRSF1) is an essential RNA-binding protein that controls posttranscriptional gene expression. We have demonstrated that SRSF1 levels are aberrantly decreased in T cells from patients with SLE and that they correlate with severe disease, yet the role of SRSF1 in T cell physiology and autoimmune disease is largely unknown. Here we show that T cell-restricted Srsf1-deficient mice develop systemic autoimmunity and lupus-nephritis. Mice exhibit increased frequencies of activated/effector T cells producing proinflammatory cytokines, and an elevated T cell activation gene signature. Mechanistically, we noted increased activity of the mechanistic target of rapamycin (mTOR) pathway and reduced expression of its repressor PTEN. The mTOR complex 1 (mTORC1) inhibitor rapamycin suppressed proinflammatory cytokine production by T cells and alleviated autoimmunity in Srsf1-deficient mice. Of direct clinical relevance, PTEN levels correlated with SRSF1 in T cells from patients with SLE, and SRSF1 overexpression rescued PTEN and suppressed mTORC1 activation and proinflammatory cytokine production. Our studies reveal the role of a previously unrecognized molecule, SRSF1, in restraining T cell activation, averting the development of autoimmune disease, and acting as a potential therapeutic target for lupus. 10.1172/JCI127949
Splicing factor SRSF6 promotes hyperplasia of sensitized skin. Jensen Mads A,Wilkinson John E,Krainer Adrian R Nature structural & molecular biology Many biological processes involve gene-expression regulation by alternative splicing. Here, we identify the splicing factor SRSF6 as a regulator of wound healing and tissue homeostasis in skin. We show that SRSF6 is a proto-oncogene frequently overexpressed in human skin cancer. Overexpressing it in transgenic mice induces hyperplasia of sensitized skin and promotes aberrant alternative splicing. We identify 139 SRSF6-target genes in skin and show that this SR-rich protein binds to alternative exons in the pre-mRNA of the extracellular-matrix protein tenascin C, thus promoting the expression of isoforms characteristic of invasive and metastatic cancer independently of cell type. SRSF6 overexpression additionally results in depletion of LGR6+ stem cells and excessive keratinocyte proliferation and response to injury. Furthermore, the effects of SRSF6 in wound healing assayed in vitro depend on the tenascin-C isoforms. Thus, abnormal SR-protein expression can perturb tissue homeostasis. 10.1038/nsmb.2756
BCLAF1 and its splicing regulator SRSF10 regulate the tumorigenic potential of colon cancer cells. Zhou Xuexia,Li Xuebing,Cheng Yuanming,Wu Wenwu,Xie Zhiqin,Xi Qiulei,Han Jun,Wu Guohao,Fang Jing,Feng Ying Nature communications Bcl-2-associated transcription factor 1 (BCLAF1) is known to be involved in multiple biological processes. Although several splice variants of BCLAF1 have been identified, little is known about how BCLAF1 splicing is regulated or the contribution of alternative splicing to its developmental functions. Here we find that inclusion of alternative exon5a was significantly increased in colorectal cancer (CRC) samples. Knockdown of the BCLAF1 protein isoform resulting from exon5a inclusion inhibited growth and that its overexpression increased tumorigenic potential. We also found that the splicing factor SRSF10 stimulates inclusion of exon5a and has growth-inducing activity. Importantly, the upregulation of SRSF10 expression observed in clinical CRC samples parallels the increased inclusion of BCLAF1 exon5a, both of which are associated with higher tumour grade. These findings identify SRSF10 as a key regulator of BCLAF1 pre-mRNA splicing and the maintenance of oncogenic features in human colon cancer cells. 10.1038/ncomms5581
Alternative splicing of the cell fate determinant Numb in hepatocellular carcinoma. Hepatology (Baltimore, Md.) UNLABELLED:The cell fate determinant Numb is aberrantly expressed in cancer. Numb is alternatively spliced, with one isoform containing a long proline-rich region (PRR(L) ) compared to the other with a short PRR (PRR(S) ). Recently, PRR(L) was reported to enhance proliferation of breast and lung cancer cells. However, the importance of Numb alternative splicing in hepatocellular carcinoma (HCC) remains unexplored. We report here that Numb PRR(L) expression is increased in HCC and associated with early recurrence and reduced overall survival after surgery. In a panel of HCC cell lines, PRR(L) generally promotes and PRR(S) suppresses proliferation, migration, invasion, and colony formation. Knockdown of PRR(S) leads to increased Akt phosphorylation and c-Myc expression, and Akt inhibition or c-Myc silencing dampens the proliferative impact of Numb PRR(S) knockdown. In the cell models explored in this study, alternative splicing of Numb PRR isoforms is coordinately regulated by the splicing factor RNA-binding Fox domain containing 2 (RbFox2) and the kinase serine/arginine protein-specific kinase 2 (SRPK2). Knockdown of the former causes accumulation of PRR(L) , while SRPK2 knockdown causes accumulation of PRR(S) . The subcellular location of SRPK2 is regulated by the molecular chaperone heat shock protein 90, and heat shock protein 90 inhibition or knockdown phenocopies SRPK2 knockdown in promoting accumulation of Numb PRR(S) . Finally, HCC cell lines that predominantly express PRR(L) are differentially sensitive to heat shock protein 90 inhibition. CONCLUSION:Alternative splicing of Numb may provide a useful prognostic biomarker in HCC and is pharmacologically tractable. 10.1002/hep.27923
RSRC1 mutation affects intellect and behaviour through aberrant splicing and transcription, downregulating IGFBP3. Perez Yonatan,Menascu Shay,Cohen Idan,Kadir Rotem,Basha Omer,Shorer Zamir,Romi Hila,Meiri Gal,Rabinski Tatiana,Ofir Rivka,Yeger-Lotem Esti,Birk Ohad S Brain : a journal of neurology RSRC1, whose polymorphism is associated with altered brain function in schizophrenia, is a member of the serine and arginine rich-related protein family. Through homozygosity mapping and whole exome sequencing we show that RSRC1 mutation causes an autosomal recessive syndrome of intellectual disability, aberrant behaviour, hypotonia and mild facial dysmorphism with normal brain MRI. Further, we show that RSRC1 is ubiquitously expressed, and that the RSRC1 mutation triggers nonsense-mediated mRNA decay of the RSRC1 transcript in patients' fibroblasts. Short hairpin RNA (shRNA)-mediated lentiviral silencing and overexpression of RSRC1 in SH-SY5Y cells demonstrated that RSRC1 has a role in alternative splicing and transcription regulation. Transcriptome profiling of RSRC1-silenced cells unravelled specific differentially expressed genes previously associated with intellectual disability, hypotonia and schizophrenia, relevant to the disease phenotype. Protein-protein interaction network modelling suggested possible intermediate interactions by which RSRC1 affects gene-specific differential expression. Patient-derived induced pluripotent stem cells, differentiated into neural progenitor cells, showed expression dynamics similar to the RSRC1-silenced SH-SY5Y model. Notably, patient neural progenitor cells had 9.6-fold downregulated expression of IGFBP3, whose brain expression is affected by MECP2, aberrant in Rett syndrome. Interestingly, Igfbp3-null mice have behavioural impairment, abnormal synaptic function and monoaminergic neurotransmission, likely correlating with the disease phenotype. 10.1093/brain/awy045
Human Tra2 proteins jointly control a CHEK1 splicing switch among alternative and constitutive target exons. Best Andrew,James Katherine,Dalgliesh Caroline,Hong Elaine,Kheirolahi-Kouhestani Mahsa,Curk Tomaz,Xu Yaobo,Danilenko Marina,Hussain Rafiq,Keavney Bernard,Wipat Anil,Klinck Roscoe,Cowell Ian G,Cheong Lee Ka,Austin Caroline A,Venables Julian P,Chabot Benoit,Santibanez Koref Mauro,Tyson-Capper Alison,Elliott David J Nature communications Alternative splicing--the production of multiple messenger RNA isoforms from a single gene--is regulated in part by RNA binding proteins. While the RBPs transformer2 alpha (Tra2α) and Tra2β have both been implicated in the regulation of alternative splicing, their relative contributions to this process are not well understood. Here we find simultaneous--but not individual--depletion of Tra2α and Tra2β induces substantial shifts in splicing of endogenous Tra2β target exons, and that both constitutive and alternative target exons are under dual Tra2α-Tra2β control. Target exons are enriched in genes associated with chromosome biology including CHEK1, which encodes a key DNA damage response protein. Dual Tra2 protein depletion reduces expression of full-length CHK1 protein, results in the accumulation of the DNA damage marker γH2AX and decreased cell viability. We conclude Tra2 proteins jointly control constitutive and alternative splicing patterns via paralog compensation to control pathways essential to the maintenance of cell viability. 10.1038/ncomms5760
SRRM4 Drives Neuroendocrine Transdifferentiation of Prostate Adenocarcinoma Under Androgen Receptor Pathway Inhibition. Li Yinan,Donmez Nilgun,Sahinalp Cenk,Xie Ning,Wang Yuwei,Xue Hui,Mo Fan,Beltran Himisha,Gleave Martin,Wang Yuzhuo,Collins Colin,Dong Xuesen European urology BACKGROUND:Neuroendocrine prostate cancer (NEPC) is an aggressive subtype of castration-resistant prostate cancer that typically does not respond to androgen receptor pathway inhibition (ARPI), and its diagnosis is increasing. OBJECTIVE:To understand how NEPC develops and to identify driver genes to inform therapy for NEPC prevention. DESIGN, SETTING, AND PARTICIPANTS:Whole-transcriptome sequencing data were extracted from prostate tumors from two independent cohorts: The Beltran cohort contained 27 adenocarcinoma and five NEPC patient samples, and the Vancouver Prostate Centre cohort contained three patient samples and nine patient-derived xenografts. INTERVENTION:A novel bioinformatics tool, comparative alternative splicing detection (COMPAS), was invented to analyze alternative RNA splicing on RNA-sequencing data. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS:COMPAS identified potential driver genes for NEPC development. Biochemical and biological validations were performed in both prostate cell and tumor models. RESULTS AND LIMITATION:More than 66% of the splice events were predicted to be regulated by the RNA splicing factor serine/arginine repetitive matrix 4 (SRRM4). In vitro and in vivo evidence confirmed that one SRRM4 target gene was the RE1 silencing transcription factor (REST), a master regulator of neurogenesis. Moreover, SRRM4 strongly stimulated adenocarcinoma cells to express NEPC biomarkers, and this effect was exacerbated by ARPI. ARPI combined with a gain of SRRM4-induced adenocarcinoma cells to assume multicellular spheroid morphology and was essential in establishing progressive NEPC xenografts. These SRRM4 actions were further enhanced by loss of function of TP53. CONCLUSIONS:SRRM4 drives NEPC progression. This knowledge may guide the development of novel therapeutics aimed at NEPC. PATIENT SUMMARY:Using next-generation RNA sequencing and our newly developed bioinformatics tool, we identified a neuroendocrine prostate cancer (NEPC)-specific RNA splicing signature that is predominantly controlled by serine/arginine repetitive matrix 4 (SRRM4). We confirmed that SRRM4 drives NEPC progression, and we propose SRRM4 as a potential therapeutic target for NEPC. 10.1016/j.eururo.2016.04.028
Alternative splicing of MBD2 supports self-renewal in human pluripotent stem cells. Cell stem cell Alternative RNA splicing (AS) regulates proteome diversity, including isoform-specific expression of several pluripotency genes. Here, we integrated global gene expression and proteomic analyses and identified a molecular signature suggesting a central role for AS in maintaining human pluripotent stem cell (hPSC) self-renewal. We demonstrate that the splicing factor SFRS2 is an OCT4 target gene required for pluripotency. SFRS2 regulates AS of the methyl-CpG binding protein MBD2, whose isoforms play opposing roles in maintenance of and reprogramming to pluripotency. Although both MDB2a and MBD2c are enriched at the OCT4 and NANOG promoters, MBD2a preferentially interacts with repressive NuRD chromatin remodeling factors and promotes hPSC differentiation, whereas overexpression of MBD2c enhances reprogramming of fibroblasts to pluripotency. The miR-301 and miR-302 families provide additional regulation by targeting SFRS2 and MDB2a. These data suggest that OCT4, SFRS2, and MBD2 participate in a positive feedback loop, regulating proteome diversity in support of hPSC self-renewal and reprogramming. 10.1016/j.stem.2014.04.002
Convergence of Acquired Mutations and Alternative Splicing of CD19 Enables Resistance to CART-19 Immunotherapy. Cancer discovery UNLABELLED:The CD19 antigen, expressed on most B-cell acute lymphoblastic leukemias (B-ALL), can be targeted with chimeric antigen receptor-armed T cells (CART-19), but relapses with epitope loss occur in 10% to 20% of pediatric responders. We detected hemizygous deletions spanning the CD19 locus and de novo frameshift and missense mutations in exon 2 of CD19 in some relapse samples. However, we also discovered alternatively spliced CD19 mRNA species, including one lacking exon 2. Pull-down/siRNA experiments identified SRSF3 as a splicing factor involved in exon 2 retention, and its levels were lower in relapsed B-ALL. Using genome editing, we demonstrated that exon 2 skipping bypasses exon 2 mutations in B-ALL cells and allows expression of the N-terminally truncated CD19 variant, which fails to trigger killing by CART-19 but partly rescues defects associated with CD19 loss. Thus, this mechanism of resistance is based on a combination of deleterious mutations and ensuing selection for alternatively spliced RNA isoforms. SIGNIFICANCE:CART-19 yield 70% response rates in patients with B-ALL, but also produce escape variants. We discovered that the underlying mechanism is the selection for preexisting alternatively spliced CD19 isoforms with the compromised CART-19 epitope. This mechanism suggests a possibility of targeting alternative CD19 ectodomains, which could improve survival of patients with B-cell neoplasms. 10.1158/2159-8290.CD-15-1020
Differential connectivity of splicing activators and repressors to the human spliceosome. Akerman Martin,Fregoso Oliver I,Das Shipra,Ruse Cristian,Jensen Mads A,Pappin Darryl J,Zhang Michael Q,Krainer Adrian R Genome biology BACKGROUND:During spliceosome assembly, protein-protein interactions (PPI) are sequentially formed and disrupted to accommodate the spatial requirements of pre-mRNA substrate recognition and catalysis. Splicing activators and repressors, such as SR proteins and hnRNPs, modulate spliceosome assembly and regulate alternative splicing. However, it remains unclear how they differentially interact with the core spliceosome to perform their functions. RESULTS:Here, we investigate the protein connectivity of SR and hnRNP proteins to the core spliceosome using probabilistic network reconstruction based on the integration of interactome and gene expression data. We validate our model by immunoprecipitation and mass spectrometry of the prototypical splicing factors SRSF1 and hnRNPA1. Network analysis reveals that a factor's properties as an activator or repressor can be predicted from its overall connectivity to the rest of the spliceosome. In addition, we discover and experimentally validate PPIs between the oncoprotein SRSF1 and members of the anti-tumor drug target SF3 complex. Our findings suggest that activators promote the formation of PPIs between spliceosomal sub-complexes, whereas repressors mostly operate through protein-RNA interactions. CONCLUSIONS:This study demonstrates that combining in-silico modeling with biochemistry can significantly advance the understanding of structure and function relationships in the human spliceosome. 10.1186/s13059-015-0682-5
The splicing factor RBM4 controls apoptosis, proliferation, and migration to suppress tumor progression. Wang Yang,Chen Dan,Qian Haili,Tsai Yihsuan S,Shao Shujuan,Liu Quentin,Dominguez Daniel,Wang Zefeng Cancer cell Splicing dysregulation is one of the molecular hallmarks of cancer. However, the underlying molecular mechanisms remain poorly defined. Here we report that the splicing factor RBM4 suppresses proliferation and migration of various cancer cells by specifically controlling cancer-related splicing. Particularly, RBM4 regulates Bcl-x splicing to induce apoptosis, and coexpression of Bcl-xL partially reverses the RBM4-mediated tumor suppression. Moreover, RBM4 antagonizes an oncogenic splicing factor, SRSF1, to inhibit mTOR activation. Strikingly, RBM4 expression is decreased dramatically in cancer patients, and the RBM4 level correlates positively with improved survival. In addition to providing mechanistic insights of cancer-related splicing dysregulation, this study establishes RBM4 as a tumor suppressor with therapeutic potential and clinical values as a prognostic factor. 10.1016/j.ccr.2014.07.010
Modulation of LMNA splicing as a strategy to treat prelamin A diseases. Lee John M,Nobumori Chika,Tu Yiping,Choi Catherine,Yang Shao H,Jung Hea-Jin,Vickers Timothy A,Rigo Frank,Bennett C Frank,Young Stephen G,Fong Loren G The Journal of clinical investigation The alternatively spliced products of LMNA, lamin C and prelamin A (the precursor to lamin A), are produced in similar amounts in most tissues and have largely redundant functions. This redundancy suggests that diseases, such as Hutchinson-Gilford progeria syndrome (HGPS), that are caused by prelamin A-specific mutations could be treated by shifting the output of LMNA more toward lamin C. Here, we investigated mechanisms that regulate LMNA mRNA alternative splicing and assessed the feasibility of reducing prelamin A expression in vivo. We identified an exon 11 antisense oligonucleotide (ASO) that increased lamin C production at the expense of prelamin A when transfected into mouse and human fibroblasts. The same ASO also reduced the expression of progerin, the mutant prelamin A protein in HGPS, in fibroblasts derived from patients with HGPS. Mechanistic studies revealed that the exon 11 sequences contain binding sites for serine/arginine-rich splicing factor 2 (SRSF2), and SRSF2 knockdown lowered lamin A production in cells and in murine tissues. Moreover, administration of the exon 11 ASO reduced lamin A expression in wild-type mice and progerin expression in an HGPS mouse model. Together, these studies identify ASO-mediated reduction of prelamin A as a potential strategy to treat prelamin A-specific diseases. 10.1172/JCI85908
Genome-wide identification of Fas/CD95 alternative splicing regulators reveals links with iron homeostasis. Tejedor J Ramón,Papasaikas Panagiotis,Valcárcel Juan Molecular cell Alternative splicing of Fas/CD95 exon 6 generates either a membrane-bound receptor that promotes, or a soluble isoform that inhibits, apoptosis. Using an automatized genome-wide siRNA screening for alternative splicing regulators of endogenous transcripts in mammalian cells, we identified 200 genes whose knockdown modulates the ratio between Fas/CD95 isoforms. These include classical splicing regulators; core spliceosome components; and factors implicated in transcription and chromatin remodeling, RNA transport, intracellular signaling, and metabolic control. Coherent effects of genes involved in iron homeostasis and pharmacological modulation of iron levels revealed a link between intracellular iron and Fas/CD95 exon 6 inclusion. A splicing regulatory network linked iron levels with reduced activity of the Zinc-finger-containing splicing regulator SRSF7, and in vivo and in vitro assays revealed that iron inhibits SRSF7 RNA binding. Our results uncover numerous links between cellular pathways and RNA processing and a mechanism by which iron homeostasis can influence alternative splicing. 10.1016/j.molcel.2014.10.029
SRSF1-Regulated Alternative Splicing in Breast Cancer. Anczuków Olga,Akerman Martin,Cléry Antoine,Wu Jie,Shen Chen,Shirole Nitin H,Raimer Amanda,Sun Shuying,Jensen Mads A,Hua Yimin,Allain Frédéric H-T,Krainer Adrian R Molecular cell Splicing factor SRSF1 is upregulated in human breast tumors, and its overexpression promotes transformation of mammary cells. Using RNA-seq, we identified SRSF1-regulated alternative splicing (AS) targets in organotypic three-dimensional MCF-10A cell cultures that mimic a context relevant to breast cancer. We identified and validated hundreds of endogenous SRSF1-regulated AS events. De novo discovery of the SRSF1 binding motif reconciled discrepancies in previous motif analyses. Using a Bayesian model, we determined positional effects of SRSF1 binding on cassette exons: binding close to the 5' splice site generally promoted exon inclusion, whereas binding near the 3' splice site promoted either exon skipping or inclusion. Finally, we identified SRSF1-regulated AS events deregulated in human tumors; overexpressing one such isoform, exon-9-included CASC4, increased acinar size and proliferation, and decreased apoptosis, partially recapitulating SRSF1's oncogenic effects. Thus, we uncovered SRSF1 positive and negative regulatory mechanisms, and oncogenic AS events that represent potential targets for therapeutics development. 10.1016/j.molcel.2015.09.005
Identification of a DNA Damage-Induced Alternative Splicing Pathway That Regulates p53 and Cellular Senescence Markers. Chen Jing,Crutchley John,Zhang Dadong,Owzar Kouros,Kastan Michael B Cancer discovery Cellular responses to DNA damage are critical determinants of cancer development and aging-associated pathogenesis. Here, we identify and characterize a DNA-damage response (DDR) pathway that regulates alternative splicing of numerous gene products, including the human tumor suppressor , and controls DNA damage-induced cellular senescence. In brief, ionizing radiation (IR) inhibits the activity of SMG1, a phosphoinositide-3-kinase-like kinase family member, reducing the binding of SMG1 to a specific region near exon 9 of p53 precursor mRNA and promoting the binding of ribosomal protein L26 (RPL26) to p53 pre-mRNA. RPL26, in turn, is required for the recruitment of the serine/arginine-rich splicing factor SRSF7 to p53 pre-mRNA and generation of alternatively spliced p53β RNA. Disruption of this pathway via selective knockout of p53β by CRISPR/Cas9 or downregulation of pathway constituents significantly reduces IR-induced senescence markers, and cells lacking p53β expression fail to transcriptionally repress negative regulators of cellular senescence and aging. We identified a new component of the DDR pathway that regulates alternative splicing of messenger RNAs, including human mRNA. Modulation of this regulatory pathway affects DNA-damage induction of cellular senescence markers. . 10.1158/2159-8290.CD-16-0908
Regulation of splicing factors by alternative splicing and NMD is conserved between kingdoms yet evolutionarily flexible. Lareau Liana F,Brenner Steven E Molecular biology and evolution Ultraconserved elements, unusually long regions of perfect sequence identity, are found in genes encoding numerous RNA-binding proteins including arginine-serine rich (SR) splicing factors. Expression of these genes is regulated via alternative splicing of the ultraconserved regions to yield mRNAs that are degraded by nonsense-mediated mRNA decay (NMD), a process termed unproductive splicing (Lareau et al. 2007; Ni et al. 2007). As all human SR genes are affected by alternative splicing and NMD, one might expect this regulation to have originated in an early SR gene and persisted as duplications expanded the SR family. But in fact, unproductive splicing of most human SR genes arose independently (Lareau et al. 2007). This paradox led us to investigate the origin and proliferation of unproductive splicing in SR genes. We demonstrate that unproductive splicing of the splicing factor SRSF5 (SRp40) is conserved among all animals and even observed in fungi; this is a rare example of alternative splicing conserved between kingdoms, yet its effect is to trigger mRNA degradation. As the gene duplicated, the ancient unproductive splicing was lost in paralogs, and distinct unproductive splicing evolved rapidly and repeatedly to take its place. SR genes have consistently employed unproductive splicing, and while it is exceptionally conserved in some of these genes, turnover in specific events among paralogs shows flexible means to the same regulatory end. 10.1093/molbev/msv002
FTO-dependent demethylation of N6-methyladenosine regulates mRNA splicing and is required for adipogenesis. Zhao Xu,Yang Ying,Sun Bao-Fa,Shi Yue,Yang Xin,Xiao Wen,Hao Ya-Juan,Ping Xiao-Li,Chen Yu-Sheng,Wang Wen-Jia,Jin Kang-Xuan,Wang Xing,Huang Chun-Min,Fu Yu,Ge Xiao-Meng,Song Shu-Hui,Jeong Hyun Seok,Yanagisawa Hiroyuki,Niu Yamei,Jia Gui-Fang,Wu Wei,Tong Wei-Min,Okamoto Akimitsu,He Chuan,Rendtlew Danielsen Jannie M,Wang Xiu-Jie,Yang Yun-Gui Cell research The role of Fat Mass and Obesity-associated protein (FTO) and its substrate N6-methyladenosine (m6A) in mRNA processing and adipogenesis remains largely unknown. We show that FTO expression and m6A levels are inversely correlated during adipogenesis. FTO depletion blocks differentiation and only catalytically active FTO restores adipogenesis. Transcriptome analyses in combination with m6A-seq revealed that gene expression and mRNA splicing of grouped genes are regulated by FTO. M6A is enriched in exonic regions flanking 5'- and 3'-splice sites, spatially overlapping with mRNA splicing regulatory serine/arginine-rich (SR) protein exonic splicing enhancer binding regions. Enhanced levels of m6A in response to FTO depletion promotes the RNA binding ability of SRSF2 protein, leading to increased inclusion of target exons. FTO controls exonic splicing of adipogenic regulatory factor RUNX1T1 by regulating m6A levels around splice sites and thereby modulates differentiation. These findings provide compelling evidence that FTO-dependent m6A demethylation functions as a novel regulatory mechanism of RNA processing and plays a critical role in the regulation of adipogenesis. 10.1038/cr.2014.151
Splicing regulator SLU7 is essential for maintaining liver homeostasis. Elizalde María,Urtasun Raquel,Azkona María,Latasa María U,Goñi Saioa,García-Irigoyen Oihane,Uriarte Iker,Segura Victor,Collantes María,Di Scala Mariana,Lujambio Amaia,Prieto Jesús,Ávila Matías A,Berasain Carmen The Journal of clinical investigation A precise equilibrium between cellular differentiation and proliferation is fundamental for tissue homeostasis. Maintaining this balance is particularly important for the liver, a highly differentiated organ with systemic metabolic functions that is endowed with unparalleled regenerative potential. Carcinogenesis in the liver develops as the result of hepatocellular de-differentiation and uncontrolled proliferation. Here, we identified SLU7, which encodes a pre-mRNA splicing regulator that is inhibited in hepatocarcinoma, as a pivotal gene for hepatocellular homeostasis. SLU7 knockdown in human liver cells and mouse liver resulted in profound changes in pre-mRNA splicing and gene expression, leading to impaired glucose and lipid metabolism, refractoriness to key metabolic hormones, and reversion to a fetal-like gene expression pattern. Additionally, loss of SLU7 also increased hepatocellular proliferation and induced a switch to a tumor-like glycolytic phenotype. Slu7 governed the splicing and/or expression of multiple genes essential for hepatocellular differentiation, including serine/arginine-rich splicing factor 3 (Srsf3) and hepatocyte nuclear factor 4α (Hnf4α), and was critical for cAMP-regulated gene transcription. Together, out data indicate that SLU7 is central regulator of hepatocyte identity and quiescence. 10.1172/JCI74382
SRSF6-regulated alternative splicing that promotes tumour progression offers a therapy target for colorectal cancer. Wan Ledong,Yu Wenying,Shen Enhui,Sun Wenjie,Liu Yuan,Kong Jianlu,Wu Yihua,Han Fengyan,Zhang Lei,Yu Tianze,Zhou Yuwei,Xie Sunzhe,Xu Enping,Zhang Honghe,Lai Maode Gut OBJECTIVE:To investigate the molecular function of splicing factor SRSF6 in colorectal cancer (CRC) progression and discover candidate chemicals for cancer therapy through targeting SRSF6. DESIGN:We performed comprehensive analysis for the expression of SRSF6 in 311 CRC samples, The Cancer Genome Atlas and Gene Expression Omnibus (GEO) database. Functional analysis of SRSF6 in CRC was performed and . SRSF6-regulated alternative splicing (AS) and its binding motif were identified by next-generation RNA-sequencing and RNA immunoprecipitation sequencing (RIP-seq), which was validated by gel shift and minigene reporter assay. ZO-1 exon23 AS was investigated to mediate the function of SRSF6 and . Based on the analysis of domain-specific role, SRSF6-targeted inhibitor was discovered by virtual screening in 4855 FDA-approved drugs and its antitumour effects were evaluated and . RESULTS:SRSF6 was frequently upregulated in CRC samples and associated with poor prognosis, which promoted proliferation and metastasis and . We identified SRSF6-regulated AS targets and discovered the SRSF6 binding motif. Particularly, SRSF6 regulates ZO-1 aberrant splicing to function as an oncogene by binding directly to its motif in the exon23. Based on the result that SRSF6 RRM2 domain plays key roles in regulating AS and biological function, indacaterol, a β2-adrenergic receptor agonist approved for chronic obstructive pulmonary disease treatment, is identified as the inhibitor of SRSF6 to suppress CRC tumourigenicity. CONCLUSIONS:SRSF6 functions the important roles in mediating CRC progression through regulating AS, and indacaterol is repositioned as an antitumour drug through targeting SRSF6. ACCESSION NUMBERS:The accession numbers for sequencing data are SRP111763 and SRP111797. 10.1136/gutjnl-2017-314983
The long non-coding RNA Gomafu is acutely regulated in response to neuronal activation and involved in schizophrenia-associated alternative splicing. Barry G,Briggs J A,Vanichkina D P,Poth E M,Beveridge N J,Ratnu V S,Nayler S P,Nones K,Hu J,Bredy T W,Nakagawa S,Rigo F,Taft R J,Cairns M J,Blackshaw S,Wolvetang E J,Mattick J S Molecular psychiatry Schizophrenia (SZ) is a complex disease characterized by impaired neuronal functioning. Although defective alternative splicing has been linked to SZ, the molecular mechanisms responsible are unknown. Additionally, there is limited understanding of the early transcriptomic responses to neuronal activation. Here, we profile these transcriptomic responses and show that long non-coding RNAs (lncRNAs) are dynamically regulated by neuronal activation, including acute downregulation of the lncRNA Gomafu, previously implicated in brain and retinal development. Moreover, we demonstrate that Gomafu binds directly to the splicing factors QKI and SRSF1 (serine/arginine-rich splicing factor 1) and dysregulation of Gomafu leads to alternative splicing patterns that resemble those observed in SZ for the archetypal SZ-associated genes DISC1 and ERBB4. Finally, we show that Gomafu is downregulated in post-mortem cortical gray matter from the superior temporal gyrus in SZ. These results functionally link activity-regulated lncRNAs and alternative splicing in neuronal function and suggest that their dysregulation may contribute to neurological disorders. 10.1038/mp.2013.45
Splicing factor SRSF1 promotes gliomagenesis via oncogenic splice-switching of MYO1B. Zhou Xuexia,Wang Run,Li Xuebing,Yu Lin,Hua Dan,Sun Cuiyun,Shi Cuijuan,Luo Wenjun,Rao Chun,Jiang Zhendong,Feng Ying,Wang Qian,Yu Shizhu The Journal of clinical investigation Abnormal alternative splicing (AS) caused by alterations to splicing factors contributes to tumor progression. Serine/arginine splicing factor 1 (SRSF1) has emerged as a key oncodriver in numerous solid tumors, leaving its roles and mechanisms largely obscure in glioma. Here, we demonstrate that SRSF1 is increased in glioma tissues and cell lines. Moreover, its expression was correlated positively with tumor grade and Ki-67 index, but inversely with patient survival. Using RNA-Seq, we comprehensively screened and identified multiple SRSF1-affected AS events. Motif analysis revealed a position-dependent modulation of AS by SRSF1 in glioma. Functionally, we verified that SRSF1 promoted cell proliferation, survival, and invasion by specifically switching the AS of the myosin IB (MYO1B) gene and facilitating the expression of the oncogenic and membrane-localized isoform, MYO1B-fl. Strikingly, MYO1B splicing was dysregulated in parallel with SRSF1 expression in gliomas and predicted the poor prognosis of the patients. Further investigation revealed that SRSF1-guided AS of the MYO1B gene increased the tumorigenic potential of glioma cells through the PDK1/AKT and PAK/LIMK pathways. Taken together, we identify SRSF1 as an important oncodriver that integrates AS control of MYO1B into promotion of gliomagenesis and represents a potential prognostic biomarker and target for glioma therapy. 10.1172/JCI120279
The Augmented R-Loop Is a Unifying Mechanism for Myelodysplastic Syndromes Induced by High-Risk Splicing Factor Mutations. Molecular cell Mutations in several general pre-mRNA splicing factors have been linked to myelodysplastic syndromes (MDSs) and solid tumors. These mutations have generally been assumed to cause disease by the resultant splicing defects, but different mutations appear to induce distinct splicing defects, raising the possibility that an alternative common mechanism is involved. Here we report a chain of events triggered by multiple splicing factor mutations, especially high-risk alleles in SRSF2 and U2AF1, including elevated R-loops, replication stress, and activation of the ataxia telangiectasia and Rad3-related protein (ATR)-Chk1 pathway. We further demonstrate that enhanced R-loops, opposite to the expectation from gained RNA binding with mutant SRSF2, result from impaired transcription pause release because the mutant protein loses its ability to extract the RNA polymerase II (Pol II) C-terminal domain (CTD) kinase-the positive transcription elongation factor complex (P-TEFb)-from the 7SK complex. Enhanced R-loops are linked to compromised proliferation of bone-marrow-derived blood progenitors, which can be partially rescued by RNase H overexpression, suggesting a direct contribution of augmented R-loops to the MDS phenotype. 10.1016/j.molcel.2017.12.029