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RNA-binding proteins (RBPs) have pivotal functions in RNA metabolism, but current methods are limited in retrieving RBP-RNA interactions within endogenous biological contexts. Here, we develop INSCRIBE (IN situ Sensitive Capture of RNA-protein Interactions in Biological Environments), circumventing the challenges through in situ RNA labeling by precisely directing a purified APOBEC1-nanobody fusion to the RBP of interest. This method enables highly specific RNA-binding site identification across a diverse range of fixed biological samples such as HEK293T cells and mouse brain tissue and accurately identifies the canonical binding motifs of RBFOX2 (UGCAUG) and TDP-43 (UGUGUG) in native cellular environments. Applicable to any RBP with available primary antibodies, INSCRIBE enables sensitive capture of RBP-RNA interactions from ultra-low input equivalent to ~5 cells. The robust, versatile, and sensitive INSCRIBE workflow is particularly beneficial for precious tissues such as clinical samples, empowering the exploration of genuine RBP-RNA interactions in RNA-related disease contexts.

Amyotrophic lateral sclerosis (ALS) is a debilitating neurodegenerative disease characterized by loss of motor neurons. Human genetic studies have linked mutations in RNA-binding proteins as causative for this disease. The hnRNPA1 protein, a known pre-mRNA splicing factor, is mutated in some ALS patients. Here, two human cell models were generated to investigate how a mutation in the C-terminal low-complexity domain (LCD) of hnRNPA1 can cause splicing changes of thousands of transcripts that collectively are linked to the DNA damage response, cilium organization, and translation. We show that the hnRNPA1 D262V mutant protein binds to new binding sites on differentially spliced transcripts from genes that are linked to ALS. We demonstrate that this ALS-linked hnRNPA1 mutation alters normal RNA-dependent protein-protein interactions. Furthermore, cells expressing this hnRNPA1 mutant exhibit a cell aggregation phenotype, markedly reduced growth rates, changes in stress granule kinetics, and aberrant growth of neuronal processes. This study provides insight into how a single amino acid mutation in a splicing factor can alter RNA splicing networks of genes linked to ALS.

Background: Dysregulated splicing events are a common phenomenon in cancer with the Serine-arginine-rich splicing factor (SRSF) family emerging as pivotal regulators of gene expression, exerting influence over constitutive and alternative splicing processes. Although aberrations in a few SRSF family members have been implicated in various cancers, the comprehensive roles of other family constituents remain underexplored. Methods: This study delves into the expression profile of the entire SRSF family (SRSF1-SRSF12) in 23 cancerous cell lines originating from diverse tissues using quantitative Real-Time PCR. Further, the transcript levels of the SRSF family were examined in oral cancer patient samples stratified into Pre-cancer (n = 15), Early cancer (n = 11), Late cancer (n = 14), and adjacent non-tumor tissues (n = 26) as controls. The results were corroborated by a parallel investigation utilizing the transcriptomics data of oral squamous cell carcinoma (OSCC) patients (n = 319) and controls (n = 35) available in The Cancer Genome Atlas (TCGA) database. Results: Our investigation reveals a notable upregulation in the expression levels of key splicing factors, namely SRSF3, SRSF9, and SRSF10 in all oral cancer cell lines (SCC-4, UM-SCC-84, CAL33, SAS-H1). Conversely, no significant associations between SRSF family members and other cancer cell lines were discerned. Further, the expression profile of the SRSF family in oral cancer patient samples revealed significant upregulation of SRSF1, SRSF3, SRSF7, SRSF9, SRSF10, and SRSF11 in patients with late-stage oral cancer compared to controls. Transcriptomics data from TCGA database demonstrated remarkable upregulation of SRSF1, SRSF4, SRSF9, SRSF10, and SRSF11 in OSCC patients. Conclusion: Collectively our results underscore the critical involvement of SRSF family members in the context of oral cancer, highlighting their potential as key players in the altered splicing dynamics associated with cancer progression. [ABSTRACT FROM AUTHOR]

The fidelity of splice site selection is critical for proper gene expression. In particular, proper recognition of 3-splice site (3SS) sequences by the spliceosome is challenging considering the low complexity of the 3SS consensus sequence YAG. Here, we show that absence of the Prp18p splicing factor results in genome-wide activation of alternative 3SS in S. cerevisiae, including highly unusual non-YAG sequences. Usage of these non-canonical 3SS in the absence of Prp18p is enhanced by upstream poly(U) tracts and by their potential to interact with the first intronic nucleoside, allowing them to dock in the spliceosome active site instead of the normal 3SS. The role of Prp18p in 3SS fidelity is facilitated by interactions with Slu7p and Prp8p, but cannot be fulfilled by Slu7p, identifying a unique role for Prp18p in 3SS fidelity. This fidelity function is synergized by the downstream proofreading activity of the Prp22p helicase, but is independent from another late splicing helicase, Prp43p. Our results show that spliceosomes exhibit remarkably relaxed 3SS sequence usage in the absence of Prp18p and identify a network of spliceosomal interactions centered on Prp18p which are required to promote the fidelity of the recognition of consensus 3SS sequences.

The mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth that stimulates macromolecule synthesis through transcription, RNA processing, and post-translational modification of metabolic enzymes. However, the mechanisms of how mTORC1 orchestrates multiple steps of gene expression programs remain unclear. Here, we identify family with sequence similarity 120A (FAM120A) as a transcription co-activator that couples transcription and splicing of de novo lipid synthesis enzymes downstream of mTORC1-serine/arginine-rich protein kinase 2 (SRPK2) signaling. The mTORC1-activated SRPK2 phosphorylates splicing factor serine/arginine-rich splicing factor 1 (SRSF1), enhancing its binding to FAM120A. FAM120A directly interacts with a lipogenic transcription factor SREBP1 at active promoters, thereby bridging the newly transcribed lipogenic genes from RNA polymerase II to the SRSF1 and U1-70K-containing RNA-splicing machinery. This mTORC1-regulated, multi-protein complex promotes efficient splicing and stability of lipogenic transcripts, resulting in fatty acid synthesis and cancer cell proliferation. These results elucidate FAM120A as a critical transcription co-factor that connects mTORC1-dependent gene regulation programs for anabolic cell growth.

BACKGROUND: Known genetic causes of congenital heart disease (CHD) explain

RNA splicing dysregulation underlies the onset and progression of cancers. In chronic lymphocytic leukemia (CLL), spliceosome mutations leading to aberrant splicing occur in ∼20% of patients. However, the mechanism for splicing defects in spliceosome-unmutated CLL cases remains elusive. Through an integrative transcriptomic and proteomic analysis, we discover that proteins involved in RNA splicing are posttranscriptionally upregulated in CLL cells, resulting in splicing dysregulation. The abundance of splicing complexes is an independent risk factor for poor prognosis. Moreover, increased splicing factor expression is highly correlated with the abundance of METTL3, an RNA methyltransferase that deposits N6-methyladenosine (m6A) on mRNA. METTL3 is essential for cell growth in vitro and in vivo and controls splicing factor protein expression in a methyltransferase-dependent manner through m6A modification-mediated ribosome recycling and decoding. Our results uncover METTL3-mediated m6A modification as a novel regulatory axis in driving splicing dysregulation and contributing to aggressive CLL.SignificanceMETTL3 controls widespread splicing factor abundance via translational control of m6A-modified mRNA, contributes to RNA splicing dysregulation and disease progression in CLL, and serves as a potential therapeutic target in aggressive CLL. See related commentary by Janin and Esteller, p. 176. This article is highlighted in the In This Issue feature, p. 171.

Pediatric acute myeloid leukemia (pAML) is typified by high relapse rates and a relative paucity of somatic DNA mutations. Although seminal studies show that splicing factor mutations and mis-splicing fuel therapy-resistant leukemia stem cell (LSC) generation in adults, splicing deregulation has not been extensively studied in pAML. Herein, we describe single-cell proteogenomics analyses, transcriptome-wide analyses of FACS-purified hematopoietic stem and progenitor cells followed by differential splicing analyses, dual-fluorescence lentiviral splicing reporter assays, and the potential of a selective splicing modulator, Rebecsinib, in pAML. Using these methods, we discover transcriptomic splicing deregulation typified by differential exon usage. In addition, we discover downregulation of splicing regulator RBFOX2 and CD47 splice isoform upregulation. Importantly, splicing deregulation in pAML induces a therapeutic vulnerability to Rebecsinib in survival, self-renewal, and lentiviral splicing reporter assays. Taken together, the detection and targeting of splicing deregulation represent a potentially clinically tractable strategy for pAML therapy.

BackgroundGlioblastoma is one of the most devastating cancer worldwide based on its locally aggressive behavior and because it cannot be cured by current therapies. Defects in alternative splicing process are frequent in cancer. Recently, we demonstrated that dysregulation of the spliceosome is directly associated with glioma development, progression, and aggressiveness.MethodsDifferent human cohorts and a dataset from different glioma mouse models were analyzed to determine the mutation frequency as well as the gene and protein expression levels between tumor and control samples of the splicing-factor-3B-subunit-1 (SF3B1), an essential and druggable spliceosome component. SF3B1 expression was also explored at the single-cell level across all cell subpopulations and transcriptomic programs. The association of SF3B1 expression with relevant clinical data (e.g., overall survival) in different human cohorts was also analyzed. Different functional (proliferation/migration/tumorspheres and colonies formation/VEGF secretion/apoptosis) and mechanistic (gene expression/signaling pathways) assays were performed in three different glioblastomas cell models (human primary cultures and cell lines) in response to SF3B1 blockade (using pladienolide B treatment). Moreover, tumor progression and formation were monitored in response to SF3B1 blockade in two preclinical xenograft glioblastoma mouse models.ResultsOur data provide novel evidence demonstrating that the splicing-factor-3B-subunit-1 (SF3B1, an essential and druggable spliceosome component) is low-frequency mutated in human gliomas (~ 1 %) but widely overexpressed in glioblastoma compared with control samples from the different human cohorts and mouse models included in the present study, wherein SF3B1 levels are associated with key molecular and clinical features (e.g., overall survival, poor prognosis and/or drug resistance). Remarkably, in vitro and in vivo blockade of SF3B1 activity with pladienolide B drastically altered multiple glioblastoma pathophysiological processes (i.e., reduction in proliferation, migration, tumorspheres formation, VEGF secretion, tumor initiation and increased apoptosis) likely by suppressing AKT/mTOR/ß-catenin pathways, and an imbalance of BCL2L1 splicing.ConclusionsTogether, we highlight SF3B1 as a potential diagnostic and prognostic biomarker and an efficient pharmacological target in glioblastoma, offering a clinically relevant opportunity worth to be explored in humans.

The spliceosome undergoes extensive rearrangements as it assembles onto precursor messenger RNAs. In the earliest assembly step, U1snRNA identifies the 5 splice site. However, U1snRNA leaves the spliceosome relatively early in assembly, and 5 splice site identity is subsequently maintained through interactions with U6snRNA, protein factor PRP8, and other components during the rearrangements that build the catalytic site. Using a forward genetic screen in Caenorhabditis elegans, we have identified suppressors of a locomotion defect caused by a 5ss mutation. Here we report three new suppressor alleles from this screen, two in PRP8 and one in SNRNP200/BRR2. mRNASeq studies of these suppressor strains indicate that they also affect specific native alternative 5ss, especially for suppressor PRP8 D1549N. A strong suppressor at the unstructured N-terminus of SNRNP200, N18K, indicates a novel role for this region. By examining distinct changes in the splicing of native genes, examining double mutants between suppressors, comparing these new suppressors to previously identified splicing suppressors from yeast, and mapping conserved suppressor residues onto cryoEM structural models of assembling human spliceosomes, we conclude that there are multiple interactions at multiple stages in spliceosome assembly responsible for maintaining the initial 5ss identified by U1snRNA for entry into the catalytic core.

SF3B1 mutations, which occur in 20% of patients with myelodysplastic syndromes (MDS), are the hallmarks of a specific MDS subtype, MDS with ringed sideroblasts (MDS-RS), which is characterized by the accumulation of erythroid precursors in the bone marrow and primarily affects the elderly population. Here, using single-cell technologies and functional validation studies of primary SF3B1-mutant MDS-RS samples, we show that SF3B1 mutations lead to the activation of the EIF2AK1 pathway in response to heme deficiency and that targeting this pathway rescues aberrant erythroid differentiation and enables the red blood cell maturation of MDS-RS erythroblasts. These data support the development of EIF2AK1 inhibitors to overcome transfusion dependency in patients with SF3B1-mutant MDS-RS with impaired red blood cell production.SignificanceMDS-RS are characterized by significant anemia. Patients with MDS-RS die from a shortage of red blood cells and the side effects of iron overload due to their constant need for transfusions. Our study has implications for the development of therapies to achieve long-lasting hematologic responses. This article is highlighted in the In This Issue feature, p. 476.

MicroRNAs (miRNAs) play an essential role in plant growth and development, and as such, their biogenesis is fine-tuned via regulation of the core microprocessor components. Here, we report that Arabidopsis AAR2, a homolog of a U5 snRNP assembly factor in yeast and humans, not only acts in splicing but also promotes miRNA biogenesis. AAR2 interacts with the microprocessor component hyponastic leaves 1 (HYL1) in the cytoplasm, nucleus, and dicing bodies. In aar2 mutants, abundance of nonphosphorylated HYL1, the active form of HYL1, and the number of HYL1-labeled dicing bodies are reduced. Primary miRNA (pri-miRNA) accumulation is compromised despite normal promoter activities of MIR genes in aar2 mutants. RNA decay assays show that the aar2-1 mutation leads to faster degradation of pri-miRNAs in a HYL1-dependent manner, which reveals a previously unknown and negative role of HYL1 in miRNA biogenesis. Taken together, our findings reveal a dual role of AAR2 in miRNA biogenesis and pre-messenger RNA splicing.

AbstractHepatitis B virus (HBV), a vaccine-avoidable infection, is a health concern worldwide, leading to liver disorders such as acute self-constraint and chronic hepatitis, liver failure, hepatic cirrhosis, and even hepatocellular carcinoma if untreated. ‘Immunogeneticprofiling’, genetic variations of the pro- and anti-inflammatory cytokines responsible for regulating the immune responses, cause person-to-person differences and impact the clinical manifestation of the disease. The current experimental–bioinformatics research was conducted to examine whether promoteric IL-18–rs187238 C > G and –rs1946518 T > G and intronic CD14–rs2569190 A > G variations are associated with chronic HBV. A total of 400 individuals (200 in each case and control group) participated in the study and were genotyped using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. The data was also assessed bioinformatics-wise for conservation, genomic transcription and splicing, and protein interactions. Findings proposed that unlike the IL-18–rs1946518 T > G and CD14–rs2569190 A > G, the IL-18–rs187238 C > G is a protector against chronic HBV (odds ratio [OR] = 0.62, 95% confidence intervals [CI]: 0.46–0.83, and p = 0.002). The TG/CC/AA, TG/CC/AG, TT/CC/AG, and GG/CC/AA combined genotypes significantly increased chronic HBV risk (p < 0.05), while the IL-18 G/T and G/G haplotypes lessened it (p < 0.05). Moreover, IL-18–rs1946518 T > G is in the protected genomic regions across mammalian species. In contrast to the IL-18–rs1946518 T > G, IL-18–rs187238 C > G is likely to create novel binding sites for transcription factors, and the CD14–rs2569190 A > G presumably changed the ribonucleic acid splicing pattern. More research on larger populations and other ethnicities is required to authenticate these results. [ABSTRACT FROM AUTHOR]

Amyotrophic lateral sclerosis (ALS) is a fatal disease characterized by aberrant alternative splicing (AS). Nuclear loss and cytoplasmic accumulation of the splicing factor TDP-43 in motor neurons (MN) are hallmarks of ALS at late stages of the disease. However, it is unknown if altered AS is present before TDP-43 pathology occurs. Here, we investigate altered AS and its origins in early stages of ALS using human induced pluripotent stem cell-derived motor neurons (MNs) from sporadic and familial ALS patients. We find high levels of the RNA-binding proteins NOVA1, NOVA2, and RBFOX2 in the insoluble protein fractions and observe that AS events in ALS-associated MNs are enriched for binding sites of these proteins. Our study points to an early disrupted function of NOVA1 that drives AS changes in a complex fashion, including events caused by a consistent loss of NOVA1 function. NOVA1 exhibits increased cytoplasmic protein levels in early stage MNs without TDP-43 pathology in ALS postmortem tissue. As nuclear TDP-43 protein level depletes, NOVA1 is reduced. Potential indications for a reduction of NOVA1 also came from mice over-expressing TDP-43 lacking its nuclear localization signal and iPSC-MN stressed with puromycin. This study highlights that additional RBP-RNA perturbations in ALS occur in parallel to TDP-43.

Rbfox1 is a multifunctional RNA-binding protein that regulates alternative splicing, transcription, mRNA stability, and translation. Rbfox1 is an important regulator of gene networks involved in neurogenesis and neuronal function. Disruption of Rbfox function has been associated with several neurodevelopmental and neuropsychiatric disorders. We have shown earlier that Rbfox1 is expressed in retinal ganglion and amacrine cells (ACs) and that its down-regulation in adult mouse retinas leads to deficiency of depth perception. In the present study, we used several markers of ACs, including gamma-aminobutyric acid (GABA), choline acetyltransferase (ChAT), neuropeptide Y (NPY), glycine transporter (GlyT1), and vesicular glutamate transporter 3 (VGlut3) to identify types of ACs that express Rbfox1. Expression of Rbfox1 was observed predominantly in GABAergic ACs located in the inner nuclear layer (INL) and ganglion cell layer (GCL). All GABAergic/cholinergic starburst ACs and virtually all NPY-positive GABAergic ACs were also Rbfox1-positive. Among glycinergic ACs, a sparse population of Rbfox1/VGlut3-positive cells was identified, indicating that Rbfox1 is expressed in a very small population of glycinergic ACs. These data contribute to our understanding about molecular differences between various types of amacrine cells and the cell-specific gene networks regulated by Rbfox1.

Serine and Arginine Rich Splicing Factor 1 (SRSF1) is a splicing factor that binds to exonic enhancers and stimulates splicing and is previously implicated with autoimmunity. Herein, we investigate the role of SRSF1 in regulating innate immune functions that are pertinent in the pathogenesis of auto-inflammatory diseases. Specifically, we show that conditional deletion of SRSF1 in mature lymphocytes resulted in higher expression of il-17a and il-17 f and an expansion of IL17A+ CD8 T cells. Mechanistically, the aberrant expression of IL-17A in SRSF1 cKO mice could not be attributed to alternative splicing of il-17a or il-17 f genes but possibly to defective CD11B+LY6C+ myeloid derived suppressor function in the spleen. Finally, meta-analysis of RNA-Seq collected from psoriasis patients demonstrate a clear correlation between SRSF1 and psoriasis that suggests a putative role of SRSF1 in IL-17A-induced psoriasis.

Molecularly targeted cancer therapy has improved outcomes for patients with cancer with targetable oncoproteins, such as mutant EGFR in lung cancer. Yet, the long-term survival of these patients remains limited, because treatment responses are typically incomplete. One potential explanation for the lack of complete and durable responses is that oncogene-driven cancers with activating mutations of EGFR often harbor additional co-occurring genetic alterations. This hypothesis remains untested for most genetic alterations that co-occur with mutant EGFR. Here, we report the functional impact of inactivating genetic alterations of the mRNA splicing factor RNA-binding motif 10 (RBM10) that co-occur with mutant EGFR. RBM10 deficiency decreased EGFR inhibitor efficacy in patient-derived EGFR-mutant tumor models. RBM10 modulated mRNA alternative splicing of the mitochondrial apoptotic regulator Bcl-x to regulate tumor cell apoptosis during treatment. Genetic inactivation of RBM10 diminished EGFR inhibitor-mediated apoptosis by decreasing the ratio of (proapoptotic) Bcl-xS to (antiapoptotic) Bcl-xL isoforms of Bcl-x. RBM10 deficiency was a biomarker of poor response to EGFR inhibitor treatment in clinical samples. Coinhibition of Bcl-xL and mutant EGFR overcame the resistance induced by RBM10 deficiency. This study sheds light on the role of co-occurring genetic alterations and on the effect of splicing factor deficiency on the modulation of sensitivity to targeted kinase inhibitor cancer therapy.

Non-core spliceosome components are essential, conserved regulators of alternative splicing. They provide concentration-dependent control of diverse pre-mRNAs. Many splicing factors direct unproductive splicing of their own pre-mRNAs through negative autoregulation. However, the impact of such feedback loops on splicing dynamics at the single-cell level remains unclear. Here, we developed a system to quantitatively analyze negative autoregulatory splicing dynamics by splicing factor SRSF1 in response to perturbations in single HEK293 cells. We show that negative autoregulatory splicing provides critical functions for gene regulation, establishing a ceiling of SRSF1 protein concentration, reducing cell-cell heterogeneity in SRSF1 levels, and buffering variation in transcription. Most important, it adapts SRSF1 splicing activity to variations in demand from other pre-mRNA substrates. A minimal mathematical model of autoregulatory splicing explains these experimentally observed features and provides values for effective biochemical parameters. These results reveal the unique functional roles that splicing negative autoregulation plays in homeostatically regulating transcriptional programs.

Amelogenin plays a crucial role in tooth enamel formation, and mutations on X-chromosomal amelogenin cause X-linked amelogenesis imperfecta (AI). Amelogenin pre–messenger RNA (mRNA) is highly alternatively spliced, and during alternative splicing, exon4 is mostly skipped, leading to the formation of a microRNA (miR-exon4) that has been suggested to function in enamel and bone formation. While delivering the functional variation of amelogenin proteins, alternative splicing of exon4 is the decisive first step to producing miR-exon4. However, the factors that regulate the splicing of exon4 are not well understood. This study aimed to investigate the association between known mutations in exon4 and exon5 of X chromosome amelogenin that causes X-linked AI, the splicing of exon4, and miR-exon4 formation. Our results showed mutations in exon4 and exon5 of the amelogenin gene, including c.120T>C, c.152C>T, c.155C>G, and c.155delC, significantly affected the splicing of exon4 and subsequent miR-exon4 production. Using an amelogenin minigene transfected in HEK-293 cells, we observed increased inclusion of exon4 in amelogenin mRNA and reduced miR-exon4 production with these mutations. In silico analysis predicted that Ser/Arg-rich RNA splicing factor (SRSF) 2 and SRSF5 were the regulatory factors for exon4 and exon5 splicing, respectively. Electrophoretic mobility shift assay confirmed that SRSF2 binds to exon4 and SRSF5 binds to exon5, and mutations in each exon can alter SRSF binding. Transfection of the amelogenin minigene to LS8 ameloblastic cells suppressed expression of the known miR-exon4 direct targets, Nfia and Prkch, related to multiple pathways. Given the mutations on the minigene, the expression of Prkch has been significantly upregulated with c.155C>G and c.155delC mutations. Together, we confirmed that exon4 splicing is critical for miR-exon4 production, and mutations causing X-linked AI in exon4 and exon5 significantly affect exon4 splicing and following miR-exon4 production. The change in miR-exon4 would be an additional etiology of enamel defects seen in some X-linked AI. [ABSTRACT FROM AUTHOR]

Purpose: Deregulation or mutation of the EZH2 gene causes various tumors, including clear cell renal cell carcinoma (ccRCC). Although several splice variants of EZH2 have been identified, little is known about how EZH2 splicing is regulated or the contribution of alternative splicing to its protumorigenic functions.Experimental Design: We conducted RT-PCR, Western blot analysis, and IHC techniques to examine EZH2 and its alternative splicing transcript expression in renal cancer tissue and renal cancer cell lines. Proliferation, migration, clonogenicity, and tumorigenicity of renal cancer cells either exhibiting knockdown of EZH2 or its splicing factor SF3B3 were assessed by CCK8, Transwell assay, and murine xenograft experiments.Results: We found that the inclusion of alternative EZH2 exon 14 was significantly increased in ccRCC samples and renal cancer cell lines. In ccRCC lines, enforced expression of EZH2Δ14 inhibited, and EZH2 promoted, cell growth, migration, proliferation, and tumorigenicity in a xenograft model. Mechanistic studies demonstrated that EZH2Δ14 isoform functions as a dominant-negative inhibitor of full-length EZH2. Coexpression of EZH2Δ14 variant with full-length EZH2 not only abrogated DAB2IP and HOXA9 suppression but also inhibited EZH2-driven tumorigenesis. Strikingly, the splicing factor SF3B3 stimulates inclusion of exon14 and has pro-proliferative activity. Importantly, the upregulation of SF3B3 expression observed in clinical ccRCC samples parallels the increased inclusion of EZH2 exon14, and the SF3B3 level is associated with higher tumor stage and poor overall survival.Conclusions: These results suggest SF3B3 as a key regulator of EZH2 pre-mRNA splicing and SF3B3 may represent a novel prognostic factor and potential therapeutic target in ccRCC. Clin Cancer Res; 23(13); 3428-41. ©2016 AACR.

Although alternative splicing is a fundamental and pervasive aspect of gene expression in higher eukaryotes, it is often omitted from single-cell studies due to quantification challenges inherent to commonly used short-read sequencing technologies. Here, we undertake the analysis of alternative splicing across numerous diverse murine cell types from two large-scale single-cell datasets-the Tabula Muris and BRAIN Initiative Cell Census Network-while accounting for understudied technical artifacts and unannotated events. We find strong and general cell-type-specific alternative splicing, complementary to total gene expression but of similar discriminatory value, and identify a large volume of novel splicing events. We specifically highlight splicing variation across different cell types in primary motor cortex neurons, bone marrow B cells, and various epithelial cells, and we show that the implicated transcripts include many genes which do not display total expression differences. To elucidate the regulation of alternative splicing, we build a custom predictive model based on splicing factor activity, recovering several known interactions while generating new hypotheses, including potential regulatory roles for novel alternative splicing events in critical genes like Khdrbs3 and Rbfox1. We make our results available using public interactive browsers to spur further exploration by the community.

Uveal melanoma (UM) is the most common intraocular tumor in adults. Recurrent mutations in BRCA1-associated protein 1 (BAP1) and splicing factor 3B subunit 1 (SF3B1) display a mutually exclusive pattern in UM, but the underlying mechanism is unknown. We show that combined BAP1 deficiency and SF3B1 hotspot mutation lead to senescence and growth arrest in human UM cells. Although p53 protein expression is induced, deletion of TP53 (encoding p53) only modestly rescues the observed senescent phenotype. UM cells with BAP1 loss or SF3B1 mutation are more sensitive to chemotherapeutic drugs compared with their isogenic parental cells. Transcriptome analysis shows that DNA-repair genes are downregulated upon co-occurrence of BAP1 deletion and SF3B1 mutation, thus leading to impaired DNA damage response and the induction of senescence. The co-occurrence of these two mutations reduces invasion of UM cells in zebrafish xenograft models and suppresses growth of melanoma xenografts in nude mice. Our findings provide a mechanistic explanation for the mutual exclusivity of BAP1 and SF3B1 mutations in human UM.

The diverse functions of WASP, the deficiency of which causes Wiskott-Aldrich syndrome (WAS), remain poorly defined. We generated three isogenic WAS models using patient induced pluripotent stem cells and genome editing. These models recapitulated WAS phenotypes and revealed that WASP deficiency causes an upregulation of numerous RNA splicing factors and widespread altered splicing. Loss of WASP binding to splicing factor gene promoters frequently leads to aberrant epigenetic activation. WASP interacts with dozens of nuclear speckle constituents and constrains SRSF2 mobility. Using an optogenetic system, we showed that WASP forms phase-separated condensates that encompasses SRSF2, nascent RNA and active Pol II. The role of WASP in gene body condensates is corroborated by ChIPseq and RIPseq. Together our data reveal that WASP is a nexus regulator of RNA splicing that controls the transcription of splicing factors epigenetically and the dynamics of the splicing machinery through liquid-liquid phase separation.

We conducted a phase I clinical trial of H3B-8800, an oral small molecule that binds Splicing Factor 3B1 (SF3B1), in patients with MDS, CMML, or AML. Among 84 enrolled patients (42 MDS, 4 CMML and 38 AML), 62 were red blood cell (RBC) transfusion dependent at study entry. Dose escalation cohorts examined two once-daily dosing regimens: schedule I (5 days on/9 days off, range of doses studied 1-40 mg, n = 65) and schedule II (21 days on/7 days off, 7-20 mg, n = 19); 27 patients received treatment for ≥180 days. The most common treatment-related, treatment-emergent adverse events included diarrhea, nausea, fatigue, and vomiting. No complete or partial responses meeting IWG criteria were observed; however, RBC transfusion free intervals >56 days were observed in nine patients who were transfusion dependent at study entry (15%). Of 15 MDS patients with missense SF3B1 mutations, five experienced RBC transfusion independence (TI). Elevated pre-treatment expression of aberrant transcripts of Transmembrane Protein 14C (TMEM14C), an SF3B1 splicing target encoding a mitochondrial porphyrin transporter, was observed in MDS patients experiencing RBC TI. In summary, H3B-8800 treatment was associated with mostly low-grade TAEs and induced RBC TI in a biomarker-defined subset of MDS.

ObjectiveThe goal of this study was to investigate the importance of central hormone-sensitive lipase (HSL) expression in the regulation of food intake and body weight in mice to clarify whether intracellular lipolysis in the mammalian hypothalamus plays a role in regulating appetite.MethodsUsing pharmacological and genetic approaches, we investigated the role of HSL in the rodent brain in the regulation of feeding and energy homeostasis under basal conditions during acute stress and high-fat diet feeding.ResultsWe found that HSL, a key enzyme in the catabolism of cellular lipid stores, is expressed in the appetite-regulating centers in the hypothalamus and is activated by acute stress through a mechanism similar to that observed in adipose tissue and skeletal muscle. Inhibition of HSL in rodent models by a synthetic ligand, global knockout, or brain-specific deletion of HSL prevents a decrease in food intake normally seen in response to acute stress and is associated with the increased expression of orexigenic peptides neuropeptide Y (NPY) and agouti-related peptide (AgRP). Increased food intake can be reversed by adeno-associated virus-mediated reintroduction of HSL in neurons of the mediobasal hypothalamus. Importantly, metabolic stress induced by a high-fat diet also enhances the hyperphagic phenotype of HSL-deficient mice. Specific deletion of HSL in the ventromedial hypothalamic nucleus (VMH) or AgRP neurons reveals that HSL in the VMH plays a role in both acute stress-induced food intake and high-fat diet-induced obesity.ConclusionsOur results indicate that HSL activity in the mediobasal hypothalamus is involved in the acute reduction in food intake during the acute stress response and sensing of a high-fat diet.

Dysregulated mTORC1 signaling alters a wide range of cellular processes, contributing to metabolic disorders and cancer. Defining the molecular details of downstream effectors is thus critical for uncovering selective therapeutic targets. We report that mTORC1 and its downstream kinase S6K enhance eIF4A/4B-mediated translation of Wilms' tumor 1-associated protein (WTAP), an adaptor for the N6-methyladenosine (m6A) RNA methyltransferase complex. This regulation is mediated by 5' UTR of WTAP mRNA that is targeted by eIF4A/4B. Single-nucleotide-resolution m6A mapping revealed that MAX dimerization protein 2 (MXD2) mRNA contains m6A, and increased m6A modification enhances its degradation. WTAP induces cMyc-MAX association by suppressing MXD2 expression, which promotes cMyc transcriptional activity and proliferation of mTORC1-activated cancer cells. These results elucidate a mechanism whereby mTORC1 stimulates oncogenic signaling via m6A RNA modification and illuminates the WTAP-MXD2-cMyc axis as a potential therapeutic target for mTORC1-driven cancers.

Small molecules that target the spliceosome SF3B complex are potent inhibitors of cancer cell growth. The compounds affect an early stage of spliceosome assembly when U2 snRNP first engages the branch point sequence of an intron. Employing an inactive herboxidiene analog (iHB) as a competitor, we investigated factors that influence inhibitor interactions with SF3B to interfere with pre-mRNA splicing in vitro. Order-of-addition experiments show that inhibitor interactions are long lasting and affected by both temperature and the presence of ATP. Our data are also consistent with the model that not all SF3B conformations observed in structural studies are conducive to productive inhibitor interactions. Notably, SF3B inhibitors do not impact an ATP-dependent rearrangement in U2 snRNP that exposes the branch binding sequence for base pairing. We also report extended structure-activity relationship analysis of the splicing inhibitor herboxidiene. We identified features of the tetrahydropyran ring that mediate its interactions with SF3B and its ability to interfere with splicing. In the context of recent structures of SF3B bound to inhibitor, our results lead us to extend the model for early spliceosome assembly and inhibitor mechanism. We postulate that interactions between a carboxylic acid substituent of herboxidiene and positively charged SF3B1 side chains in the inhibitor binding channel are needed to maintain inhibitor occupancy while counteracting the SF3B transition to a closed state that is required for stable U2 snRNP interactions with the intron.

Autosomal-recessive cerebellar hypoplasia and ataxia constitute a group of heterogeneous brain disorders caused by disruption of several fundamental cellular processes. Here, we identified 10 families showing a neurodegenerative condition involving pontocerebellar hypoplasia with microcephaly (PCHM). Patients harbored biallelic mutations in genes encoding the spliceosome components Peptidyl-Prolyl Isomerase Like-1 (PPIL1) or Pre-RNA Processing-17 (PRP17). Mouse knockouts of either gene were lethal in early embryogenesis, whereas PPIL1 patient mutation knockin mice showed neuron-specific apoptosis. Loss of either protein affected splicing integrity, predominantly affecting short and high GC-content introns and genes involved in brain disorders. PPIL1 and PRP17 form an active isomerase-substrate interaction, but we found that isomerase activity is not critical for function. Thus, we establish disrupted splicing integrity and "major spliceosome-opathies" as a new mechanism underlying PCHM and neurodegeneration and uncover a non-enzymatic function of a spliceosomal proline isomerase.

The decoy exon model has been proposed to regulate a subset of intron retention (IR) events involving predominantly larger introns (>1 kb). Splicing reporter studies have shown that decoy splice sites are essential for activity, suggesting that decoys act by engaging intron-terminal splice sites and competing with cross-intron interactions required for intron excision. The decoy model predicts that antisense oligonucleotides may be able to block decoy splice sites in endogenous pre-mRNA, thereby reducing IR and increasing productive gene expression. Indeed, we now demonstrate that targeting a decoy 5' splice site in the O-GlcNAc transferase (OGT) gene reduced IR from ∼80% to ∼20% in primary human erythroblasts, accompanied by increases in spliced OGT RNA and OGT protein expression. The remaining OGT IR was refractory to antisense treatment and might be mediated by independent mechanism(s). In contrast, other retained introns were strongly dependent on decoy function, since antisense targeting of decoy 5' splice sites greatly reduced (SNRNP70) or nearly eliminated (SF3B1) IR in two widely expressed splicing factors, and also greatly reduced IR in transcripts encoding the erythroid-specific structural protein, α-spectrin (SPTA1). These results show that modulating decoy exon function can dramatically alter IR and suggest that dynamic regulation of decoy exons could be a mechanism to fine-tune gene expression post-transcriptionally in many cell types.

The genetic basis and corresponding clinical relevance of prolactinomas remain poorly understood. Here, we perform whole genome sequencing (WGS) on 21 patients with prolactinomas to detect somatic mutations and then validate the mutations with digital polymerase chain reaction (PCR) analysis of tissue samples from 227 prolactinomas. We identify the same hotspot somatic mutation in splicing factor 3 subunit B1 (SF3B1R625H) in 19.8% of prolactinomas. These patients with mutant prolactinomas display higher prolactin (PRL) levels (p = 0.02) and shorter progression-free survival (PFS) (p = 0.02) compared to patients without the mutation. Moreover, we identify that the SF3B1R625H mutation causes aberrant splicing of estrogen related receptor gamma (ESRRG), which results in stronger binding of pituitary-specific positive transcription factor 1 (Pit-1), leading to excessive PRL secretion. Thus our study validates an important mutation and elucidates a potential mechanism underlying the pathogenesis of prolactinomas that may lead to the development of targeted therapeutics.

While splicing changes caused by somatic mutations in SF3B1 are known, identifying full-length isoform changes may better elucidate the functional consequences of these mutations. We report nanopore sequencing of full-length cDNA from CLL samples with and without SF3B1 mutation, as well as normal B cell samples, giving a total of 149 million pass reads. We present FLAIR (Full-Length Alternative Isoform analysis of RNA), a computational workflow to identify high-confidence transcripts, perform differential splicing event analysis, and differential isoform analysis. Using nanopore reads, we demonstrate differential 3' splice site changes associated with SF3B1 mutation, agreeing with previous studies. We also observe a strong downregulation of intron retention events associated with SF3B1 mutation. Full-length transcript analysis links multiple alternative splicing events together and allows for better estimates of the abundance of productive versus unproductive isoforms. Our work demonstrates the potential utility of nanopore sequencing for cancer and splicing research.

A vast amount of public RNA-sequencing datasets have been generated and used widely to study transcriptome mechanisms. These data offer precious opportunity for advancing biological research in transcriptome studies such as alternative splicing. We report the first large-scale integrated analysis of RNA-Seq data of splicing factors for systematically identifying key factors in diseases and biological processes. We analyzed 1,321 RNA-Seq libraries of various mouse tissues and cell lines, comprising more than 6.6 TB sequences from 75 independent studies that experimentally manipulated 56 splicing factors. Using these data, RNA splicing signatures and gene expression signatures were computed, and signature comparison analysis identified a list of key splicing factors in Rett syndrome and cold-induced thermogenesis. We show that cold-induced RNA-binding proteins rescue the neurite outgrowth defects in Rett syndrome using neuronal morphology analysis, and we also reveal that SRSF1 and PTBP1 are required for energy expenditure in adipocytes using metabolic flux analysis. Our study provides an integrated analysis for identifying key factors in diseases and biological processes and highlights the importance of public data resources for identifying hypotheses for experimental testing.

Rbfox proteins regulate alternative splicing, mRNA stability and translation. These proteins are involved in neurogenesis and have been associated with various neurological conditions. Here, we analyzed Rbfox2 expression in adult and developing mouse retinas and the effect of its downregulation on visual function and retinal transcriptome. In adult rodents, Rbfox2 is expressed in all retinal ganglion cell (RGC) subtypes, horizontal cells, as well as GABAergic amacrine cells (ACs). Among GABAergic AC subtypes, Rbfox2 was colocalized with cholinergic starburst ACs, NPY (neuropeptide Y)- and EBF1 (early B-cell factor 1)-positive ACs. In differentiating retinal cells, Rbfox2 expression was observed as early as E12 and, unlike Rbfox1, which changes its subcellular localization from cytoplasmic to predominantly nuclear at around P0, Rbfox2 remains nuclear throughout retinal development. Rbfox2 knockout in adult animals had no detectable effect on retinal gross morphology. However, the visual cliff test revealed a significant abnormality in the depth perception of Rbfox2-deficient animals. Gene set enrichment analysis identified genes regulating the RNA metabolic process as a top enriched class of genes in Rbfox2-deficient retinas. Pathway analysis of the top 100 differentially expressed genes has identified Rbfox2-regulated genes associated with circadian rhythm and entrainment, glutamatergic/cholinergic/dopaminergic synaptic function, calcium and PI3K-AKT signaling.

The 3' UTR (UTR) of human mRNAs plays a critical role in controlling protein expression and function. Importantly, 3' UTRs of human messages are not invariant for each gene but rather are shaped by alternative polyadenylation (APA) in a cell state-dependent manner, including in response to T cell activation. However, the proteins and mechanisms driving APA regulation remain poorly understood. Here we show that the RNA-binding protein CELF2 controls APA of its own message in a signal-dependent manner by competing with core enhancers of the polyadenylation machinery for binding to RNA. We further show that CELF2 binding overlaps with APA enhancers transcriptome-wide, and almost half of 3' UTRs that undergo T cell signaling-induced APA are regulated in a CELF2-dependent manner. These studies thus reveal CELF2 to be a critical regulator of 3' UTR identity in T cells and demonstrate an additional mechanism for CELF2 in regulating polyadenylation site choice.

Myelodysplastic syndromes (MDS) with ring sideroblasts are hematopoietic stem cell disorders with erythroid dysplasia and mutations in the SF3B1 splicing factor gene. Patients with MDS with SF3B1 mutations often accumulate excessive tissue iron, even in the absence of transfusions, but the mechanisms that are responsible for their parenchymal iron overload are unknown. Body iron content, tissue distribution, and the supply of iron for erythropoiesis are controlled by the hormone hepcidin, which is regulated by erythroblasts through secretion of the erythroid hormone erythroferrone (ERFE). Here, we identified an alternative ERFE transcript in patients with MDS with the SF3B1 mutation. Induction of this ERFE transcript in primary SF3B1-mutated bone marrow erythroblasts generated a variant protein that maintained the capacity to suppress hepcidin transcription. Plasma concentrations of ERFE were higher in patients with MDS with an SF3B1 gene mutation than in patients with SF3B1 wild-type MDS. Thus, hepcidin suppression by a variant ERFE is likely responsible for the increased iron loading in patients with SF3B1-mutated MDS, suggesting that ERFE could be targeted to prevent iron-mediated toxicity. The expression of the variant ERFE transcript that was restricted to SF3B1-mutated erythroblasts decreased in lenalidomide-responsive anemic patients, identifying variant ERFE as a specific biomarker of clonal erythropoiesis.

Heart performance relies on highly coordinated excitation-contraction (EC) coupling, and defects in this critical process may be exacerbated by additional genetic defects and/or environmental insults to cause eventual heart failure. Here we report a regulatory pathway consisting of the RNA binding protein RBFox2, a stress-induced microRNA miR-34a, and the essential EC coupler JPH2. In this pathway, initial cardiac defects diminish RBFox2 expression, which induces transcriptional repression of miR-34a, and elevated miR-34a targets Jph2 to impair EC coupling, which further manifests heart dysfunction, leading to progressive heart failure. The key contribution of miR-34a to this process is further established by administrating its mimic, which is sufficient to induce cardiac defects, and by using its antagomir to alleviate RBFox2 depletion-induced heart dysfunction. These findings elucidate a potential feed-forward mechanism to account for a critical transition to cardiac decompensation and suggest a potential therapeutic avenue against heart failure.

In this study, we investigated low-frequency and rare variants associated with blood pressure (BP) by focusing on a linkage region on chromosome 16p13. We used whole genome sequencing (WGS) data obtained through the NHLBI Trans-Omics for Precision Medicine (TOPMed) program on 395 Cleveland Family Study (CFS) European Americans (CFS-EA). By analyzing functional coding variants and non-coding rare variants with CADD score > 10 residing within the chromosomal region in families with linkage evidence, we observed 25 genes with nominal statistical evidence (burden or SKAT p

SF3B1 is recurrently mutated in chronic lymphocytic leukemia (CLL), but its role in the pathogenesis of CLL remains elusive. Here, we show that conditional expression of Sf3b1-K700E mutation in mouse B cells disrupts pre-mRNA splicing, alters cell development, and induces a state of cellular senescence. Combination with Atm deletion leads to the overcoming of cellular senescence and the development of CLL-like disease in elderly mice. These CLL-like cells show genome instability and dysregulation of multiple CLL-associated cellular processes, including deregulated B cell receptor signaling, which we also identified in human CLL cases. Notably, human CLLs harboring SF3B1 mutations exhibit altered response to BTK inhibition. Our murine model of CLL thus provides insights into human CLL disease mechanisms and treatment.

The spliceosome accurately promotes precursor messenger-RNA splicing by recognizing specific noncoding intronic tracts including the branch point sequence (BPS) and the 3'-splice-site (3'SS). Mutations of Hsh155 (yeast)/SF3B1 (human), which is a protein of the SF3b factor involved in BPS recognition and induces altered BPS binding and 3'SS selection, lead to mis-spliced mRNA transcripts. Although these mutations recur in hematologic malignancies, the mechanism by which they change gene expression remains unclear. In this study, multi-microsecond-long molecular-dynamics simulations of eighth distinct ∼700,000 atom models of the spliceosome Bact complex, and gene sequencing of SF3B1, disclose that these carcinogenic isoforms destabilize intron binding and/or affect the functional dynamics of Hsh155/SF3B1 only when binding non-consensus BPSs, as opposed to the non-pathogenic variants newly annotated here. This pinpoints a cross-talk between the distal Hsh155 mutation and BPS recognition sites. Our outcomes unprecedentedly contribute to elucidating the principles of pre-mRNA recognition, which provides critical insights on the mechanism underlying constitutive/alternative/aberrant splicing.

Myeloid malignancies, including myelodysplastic syndromes, chronic myelomonocytic leukemia, and acute myeloid leukemia, are characterized by abnormal proliferation and differentiation of hematopoietic stem and progenitor cells (HSPCs). Reports on analysis of bone marrow samples from patients have revealed a high incidence of mutations in splicing factors in early stem and progenitor cell clones, but the mechanisms underlying transformation of HSPCs harboring these mutations remain unknown. Using ex vivo cultures of primary human CD34+ cells as a model, we find that mutations in splicing factors SRSF2 and U2AF1 exert distinct effects on proliferation and differentiation of HSPCs. SRSF2 mutations cause a dramatic inhibition of proliferation via a G2-M phase arrest and induction of apoptosis. U2AF1 mutations, conversely, do not significantly affect proliferation. Mutations in both SRSF2 and U2AF1 cause abnormal differentiation by skewing granulo-monocytic differentiation toward monocytes but elicit diverse effects on megakaryo-erythroid differentiation. The SRSF2 mutations skew differentiation toward megakaryocytes whereas U2AF1 mutations cause an increase in the erythroid cell populations. These distinct functional consequences indicate that SRSF2 and U2AF1 mutations have cell context-specific effects and that the generation of myeloid disease phenotype by mutations in the genes coding these two proteins likely involves different intracellular mechanisms. Stem Cells 2018;36:1663-1675.

The identification of targetable vulnerabilities in the context of therapeutic resistance is a key challenge in cancer treatment. We detected pervasive aberrant splicing as a characteristic feature of chronic lymphocytic leukemia (CLL), irrespective of splicing factor mutation status, which was associated with sensitivity to the spliceosome modulator, E7107. Splicing modulation affected CLL survival pathways, including members of the B cell lymphoma-2 (BCL2) family of proteins, remodeling antiapoptotic dependencies of human and murine CLL cells. E7107 treatment decreased myeloid cell leukemia-1 (MCL1) dependence and increased BCL2 dependence, sensitizing primary human CLL cells and venetoclax-resistant CLL-like cells from an Eμ-TCL1-based adoptive transfer murine model to treatment with the BCL2 inhibitor venetoclax. Our data provide preclinical rationale to support the combination of venetoclax with splicing modulators to reprogram apoptotic dependencies in CLL for treating venetoclax-resistant CLL cases.

During terminal erythropoiesis, the splicing machinery in differentiating erythroblasts executes a robust intron retention (IR) program that impacts expression of hundreds of genes. We studied IR mechanisms in the SF3B1 splicing factor gene, which expresses ∼50% of its transcripts in late erythroblasts as a nuclear isoform that retains intron 4. RNA-seq analysis of nonsense-mediated decay (NMD)-inhibited cells revealed previously undescribed splice junctions, rare or not detected in normal cells, that connect constitutive exons 4 and 5 to highly conserved cryptic cassette exons within the intron. Minigene splicing reporter assays showed that these cassettes promote IR. Genome-wide analysis of splice junction reads demonstrated that cryptic noncoding cassettes are much more common in large (>1 kb) retained introns than they are in small retained introns or in nonretained introns. Functional assays showed that heterologous cassettes can promote retention of intron 4 in the SF3B1 splicing reporter. Although many of these cryptic exons were spliced inefficiently, they exhibited substantial binding of U2AF1 and U2AF2 adjacent to their splice acceptor sites. We propose that these exons function as decoys that engage the intron-terminal splice sites, thereby blocking cross-intron interactions required for excision. Developmental regulation of decoy function underlies a major component of the erythroblast IR program.

Alternative pre-mRNA splicing (AS) greatly diversifies metazoan transcriptomes and proteomes and is crucial for gene regulation. Current computational analysis methods of AS from Illumina RNA-sequencing data rely on preannotated libraries of known spliced transcripts, which hinders AS analysis with poorly annotated genomes and can further mask unknown AS patterns. To address this critical bioinformatics problem, we developed a method called the junction usage model (JUM) that uses a bottom-up approach to identify, analyze, and quantitate global AS profiles without any prior transcriptome annotations. JUM accurately reports global AS changes in terms of the five conventional AS patterns and an additional "composite" category composed of inseparable combinations of conventional patterns. JUM stringently classifies the difficult and disease-relevant pattern of intron retention (IR), reducing the false positive rate of IR detection commonly seen in other annotation-based methods to near-negligible rates. When analyzing AS in RNA samples derived from Drosophila heads, human tumors, and human cell lines bearing cancer-associated splicing factor mutations, JUM consistently identified approximately twice the number of novel AS events missed by other methods. Computational simulations showed JUM exhibits a 1.2 to 4.8 times higher true positive rate at a fixed cutoff of 5% false discovery rate. In summary, JUM provides a framework and improved method that removes the necessity for transcriptome annotations and enables the detection, analysis, and quantification of AS patterns in complex metazoan transcriptomes with superior accuracy.

N1-methyladenosine (m1A) is an important post-transcriptional modification in RNA; however, the exact biological role of m1A remains to be determined. By employing a quantitative proteomics method, we identified multiple putative protein readers of m1A in RNA, including several YTH domain family proteins. We showed that YTHDF1-3 and YTHDC1, but not YTHDC2, could bind directly to m1A in RNA. We also found that Trp432 in YTHDF2, a conserved residue in the hydrophobic pocket of the YTH domain that is necessary for its binding to N6-methyladenosine (m6A), is required for its recognition of m1A. An analysis of previously published data revealed transcriptome-wide colocalization of YTH domain-containing proteins and m1A sites in HeLa cells, suggesting that YTH domain-containing proteins can bind to m1A in cells. Together, our results uncovered YTH domain-containing proteins as readers for m1A in RNA and provided new insight into the functions of m1A in RNA biology.

Tools to understand how the spliceosome functions in vivo have lagged behind advances in the structural biology of the spliceosome. Here, methods are described to globally profile spliceosome-bound pre-mRNA, intermediates, and spliced mRNA at nucleotide resolution. These tools are applied to three yeast species that span 600 million years of evolution. The sensitivity of the approach enables the detection of canonical and non-canonical events, including interrupted, recursive, and nested splicing. This application of statistical modeling uncovers independent roles for the size and position of the intron and the number of introns per transcript in substrate progression through the two catalytic stages. These include species-specific inputs suggestive of spliceosome-transcriptome coevolution. Further investigations reveal the ATP-dependent discard of numerous endogenous substrates after spliceosome assembly in vivo and connect this discard to intron retention, a form of splicing regulation. Spliceosome profiling is a quantitative, generalizable global technology used to investigate an RNP central to eukaryotic gene expression.

Hotspot mutations in splicing factor genes have been recently reported at high frequency in hematological malignancies, suggesting the importance of RNA splicing in cancer. We analyzed whole-exome sequencing data across 33 tumor types in The Cancer Genome Atlas (TCGA), and we identified 119 splicing factor genes with significant non-silent mutation patterns, including mutation over-representation, recurrent loss of function (tumor suppressor-like), or hotspot mutation profile (oncogene-like). Furthermore, RNA sequencing analysis revealed altered splicing events associated with selected splicing factor mutations. In addition, we were able to identify common gene pathway profiles associated with the presence of these mutations. Our analysis suggests that somatic alteration of genes involved in the RNA-splicing process is common in cancer and may represent an underappreciated hallmark of tumorigenesis.

Dysfunction of the neuronal RNA binding protein RBFOX1 has been linked to epilepsy and autism spectrum disorders. Rbfox1 loss in mice leads to neuronal hyper-excitability and seizures, but the physiological basis for this is unknown. We identify the vSNARE protein Vamp1 as a major Rbfox1 target. Vamp1 is strongly downregulated in Rbfox1 Nes-cKO mice due to loss of 3' UTR binding by RBFOX1. Cytoplasmic Rbfox1 stimulates Vamp1 expression in part by blocking microRNA-9. We find that Vamp1 is specifically expressed in inhibitory neurons, and that both Vamp1 knockdown and Rbfox1 loss lead to decreased inhibitory synaptic transmission and E/I imbalance. Re-expression of Vamp1 selectively within interneurons rescues the electrophysiological changes in the Rbfox1 cKO, indicating that Vamp1 loss is a major contributor to the Rbfox1 Nes-cKO phenotype. The regulation of interneuron-specific Vamp1 by Rbfox1 provides a paradigm for broadly expressed RNA-binding proteins performing specialized functions in defined neuronal subtypes.

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.

Rbfox1 is a splicing regulator that has been associated with various neurological conditions such as autism spectrum disorder, mental retardation, epilepsy, attention-deficit/hyperactivity disorder and schizophrenia. We show that in adult rodent retinas, Rbfox1 is expressed in all types of retinal ganglion cells (RGCs) and in certain subsets of amacrine cells (ACs), within the inner nuclear (INL) and ganglion cell (GCL) layers. In the INL, all Rbfox1-positive cells were colocalized with GABAergic ACs, however not all GABAergic ACs were immunostained for Rbfox1. In the GCL, a vast majority of GABAergic dACs were Rbfox1-immunopositive. Furthermore, all cholinergic starburst ACs (SACs) in the INL (type a) and in the GCL (type b) were Rbfox1 positive. The expression of Rbfox1 in the retina significantly overlapped with expression of Rbfox2, another member of Rbfox family of proteins. Rbfox2, in addition to RGCs and ACs, was also expressed in horizontal cells. In developing retinas at E12 and E15, Rbfox1 is localized to the cytoplasm of differentiating RGCs and ACs. Between P0 and P5, Rbfox1 subcellular localization switched from cytoplasmic to predominantly nuclear. Downregulation of Rbfox1 in adult Rbfox1loxP/loxP mice had no detectable effect on retinal gross morphology. However, the visual cliff test revealed marked abnormalities of depth perception of these animals. RNA sequencing of retinal transcriptomes of control and Rbfox1 knockout animals identified a number of Rbfox1-regulated genes that are involved in establishing neuronal circuits and synaptic transmission, including Vamp1, Vamp2, Snap25, Trak2, and Slc1A7, suggesting the role of Rbfox1 in facilitating synaptic communications between ACs and RGCs.

The spliceosome undergoes dramatic changes in a splicing cycle. Structures of B, Bact, C, C*, and intron lariat spliceosome complexes revealed mechanisms of 5'-splice site (ss) recognition, branching, and intron release, but lacked information on 3'-ss recognition, exon ligation, and exon release. Here we report a cryo-electron microscopy structure of the postcatalytic P complex at 3.3-angstrom resolution, revealing that the 3' ss is mainly recognized through non-Watson-Crick base pairing with the 5' ss and branch point. Furthermore, one or more unidentified proteins become stably associated with the P complex, securing the 3' exon and potentially regulating activity of the helicase Prp22. Prp22 binds nucleotides 15 to 21 in the 3' exon, enabling it to pull the intron-exon or ligated exons in a 3' to 5' direction to achieve 3'-ss proofreading or exon release, respectively.