Identification of splice regulators of fibronectin-EIIIA and EIIIB by direct measurement of exon usage in a flow-cytometry based CRISPR screen

The extracellular matrix protein fibronectin (FN) is alternatively spliced in a variety of inflammatory conditions, resulting in increased inclusion of alternative exons EIIIA and EIIIB. Inclusion of these exons affects fibril formation, fibrosis, and inflammation. To define upstream regulators of alternative splicing in FN, we have developed an in vitro flow-cytometry based assay, using RNA-binding probes to determine alternative exon inclusion level in aortic endothelial cells. This approach allows us to detect exon inclusion in the primary transcripts themselves, rather than in surrogate splicing reporters. We validated this assay in cells with and without FN-EIIIA and -EIIIB expression. In a small-scale CRISPR KO screen of candidate regulatory splice factors, we successfully detected known regulators of EIIIA and EIIIB splicing, and detected several novel regulators. Finally, we show the potential in this approach to broadly interrogate upstream signaling pathways in aortic endothelial cells with a genome-wide CRISPR-KO screen, implicating the TNFalpha and RIG-I-like signaling pathways and genes involved in the regulation of fibrotic responses. Thus, we provide a novel means to screen the regulation of splicing of endogenous transcripts, and predict novel pathways in the regulation of FN-EIIIA inclusion.

Identification of Splice Regulators of Fibronectin-EIIIA and EIIIB by Direct Measurement of Exon Usage in a Flow-Cytometry Based CRISPR Screen

The extracellular matrix protein fibronectin (FN) is alternatively spliced in a variety of inflammatory conditions, resulting in increased inclusion of alternative exons EIIIA and EIIIB. Inclusion of these exons affects fibril formation, fibrosis, and inflammation. To define upstream regulators of alternative splicing in FN, we have developed an in vitro flow-cytometry based assay, using RNA-binding probes to determine alternative exon inclusion level in aortic endothelial cells. This approach allows us to detect exon inclusion in the primary transcripts themselves, rather than in surrogate splicing reporters. We validated this assay in cells with and without FN-EIIIA and –EIIIB expression. In a small-scale CRISPR KO screen of candidate regulatory splice factors, we successfully detected known regulators of EIIIA and EIIIB splicing, and detected several novel regulators. Finally, we show the potential in this approach to broadly interrogate upstream signaling pathways in aortic endothelial cells with a genome-wide CRISPR-KO screen, implicating the TNFalpha and RIG-I-like signaling pathways and genes involved in the regulation of fibrotic responses. Thus, we provide a novel means to screen the regulation of splicing of endogenous transcripts, and predict novel pathways in the regulation of FN-EIIIA inclusion.

TDP-43 represses cryptic exon inclusion in FTD/ALS gene UNC13A

A hallmark pathological feature of neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is the depletion of RNA-binding protein TDP-43 from the nucleus of neurons in the brain and spinal cord. A major function of TDP-43 is as a repressor of cryptic exon inclusion during RNA splicing. Single nucleotide polymorphisms (SNPs) in UNC13A are among the strongest genome-wide association study (GWAS) hits associated with FTD/ALS in humans, but how those variants increase risk for disease is unknown. Here we show that TDP-43 represses a cryptic exon splicing event in UNC13A. Loss of TDP-43 from the nucleus in human brain, neuronal cell lines, and iPSC-derived motor neurons resulted in the inclusion of a cryptic exon in UNC13A mRNA and reduced UNC13A protein expression. Remarkably, the top variants associated with FTD/ALS risk in humans are located in the cryptic exon harboring intron itself and we show that they increase UNC13A cryptic exon splicing in the face of TDP-43 dysfunction. Together, our data provide a direct functional link between one of the strongest genetic risk factors for FTD/ALS (UNC13A genetic variants) and loss of TDP-43 function.

Mutations primarily alter the inclusion of alternatively spliced exons

Genetic analyses and systematic mutagenesis have revealed that synonymous, non-synonymous and intronic mutations frequently alter the inclusion levels of alternatively spliced exons, consistent with the concept that altered splicing might be a common mechanism by which mutations cause disease. However, most exons expressed in any cell are highly-included in mature mRNAs. Here, by performing deep mutagenesis of highly-included exons and by analysing the association between genome sequence variation and exon inclusion across the transcriptome, we report that mutations only very rarely alter the inclusion of highly-included exons. This is true for both exonic and intronic mutations as well as for perturbations in trans. Therefore, mutations that affect splicing are not evenly distributed across primary transcripts but are focussed in and around alternatively spliced exons with intermediate inclusion levels. These results provide a resource for prioritising synonymous and other variants as disease-causing mutations.

Mutations primarily alter the inclusion of alternatively spliced exons

Genetic analyses and systematic mutagenesis have revealed that synonymous, non-synonymous and intronic mutations frequently alter the inclusion levels of alternatively spliced exons, consistent with the concept that altered splicing might be a common mechanism by which mutations cause disease. However, most exons expressed in any cell are highly-included in mature mRNAs. Here, by performing deep mutagenesis of highly-included exons and by analysing the association between genome sequence variation and exon inclusion across the transcriptome, we report that mutations only very rarely alter the inclusion of highly-included exons. This is true for both exonic and intronic mutations as well as for perturbations in trans. Therefore, mutations that affect splicing are not evenly distributed across primary transcripts but are focussed in and around alternatively spliced exons with intermediate inclusion levels. These results provide a resource for prioritising synonymous and other variants as disease-causing mutations.

Aberrant regulation of a poison exon caused by a non-coding variant in Scn1a-associated epileptic encephalopathy

Dravet syndrome (DS) is a developmental and epileptic encephalopathy that results from mutations in the Nav1.1 sodium channel encoded by SCN1A. Most known DS-causing mutations are in coding regions of SCN1A, but we recently identified several disease-associated SCN1A mutations in intron 20 that are within or near to a cryptic and evolutionarily conserved “poison” exon, 20N, whose inclusion leads to transcript degradation. However, it is not clear how these intron 20 variants alter SCN1A transcript processing or DS pathophysiology in an organismal context, nor is it clear how exon 20N is regulated in a tissue-specific and developmental context. We address those questions here by generating an animal model of our index case, NM_006920.4(SCN1A):c.3969+2451G>C, using gene editing to create the orthologous mutation in laboratory mice. Scn1a heterozygous knock-in (+/KI) mice exhibited an ~50% reduction in brain Scn1a mRNA and Nav1.1 protein levels, together with characteristics observed in other DS mouse models, including premature mortality, seizures, and hyperactivity. In brain tissue from adult Scn1a +/+ animals, quantitative RT-PCR assays indicated that ~1% of Scn1a mRNA included exon 20N, while brain tissue from Scn1a +/KI mice exhibited an ~5-fold increase in the extent of exon 20N inclusion. We investigated the extent of exon 20N inclusion in brain during normal fetal development in RNA-seq data and discovered that levels of inclusion were ~70% at E14.5, declining progressively to ~10% postnatally. A similar pattern exists for the homologous sodium channel Nav1.6, encoded by Scn8a. For both genes, there is an inverse relationship between the level of functional transcript and the extent of poison exon inclusion. Taken together, our findings suggest that poison exon usage by Scn1a and Scn8a is a strategy to regulate channel expression during normal brain development, and that mutations recapitulating a fetal-like pattern of splicing cause reduced channel expression and epileptic encephalopathy.Author SummaryDravet syndrome (DS) is a neurological disorder affecting approximately 1:15,700 Americans[1]. While most patients have a mutation in the SCN1A gene encoding Nav1.1 sodium channels, about 20% do not have a mutation identified by exome sequencing. Recently, we identified variants in intron 20N, a noncoding region of SCN1A, in some DS patients [2]. We predicted that these variants alter SCN1A transcript processing, decrease Nav1.1 function, and lead to DS pathophysiology via inclusion of exon 20N, a “poison” exon that leads to a premature stop codon. In this study, we generated a knock-in mouse model, Scn1a+/KI, of one of these variants, NM_006920.4(SCN1A):c.3969+2451G>C, which resides in a genomic region that is extremely conserved across vertebrate species. We found that Scn1a+/KI mice have reduced levels of Scn1a transcript and Nav1.1 protein and develop DS-related phenotypes. Consistent with the poison exon hypothesis, transcripts from brains of Scn1a+/KI mice showed elevated rates of Scn1a exon 20N inclusion. Since Scn1a expression in the brain is regulated developmentally, we next explored the developmental relationship between exon 20N inclusion and Scn1a expression. During normal embryogenesis, when Scn1a expression was low, exon 20N inclusion was high; postnatally, as Scn1a expression increased, there was a corresponding decrease in exon 20N usage. Expression of another voltage-gated sodium channel transcript, Scn8a (Nav1.6), was similarly regulated, with inclusion of a poison exon termed as 18N early in development when Scn8a expression was low, followed by a postnatal decrease in exon 18N inclusion and corresponding increase in Scn8a expression. Together, these data demonstrate that poison exon inclusion is a conserved mechanism to control sodium channel expression in the brain, and that an intronic mutation that disrupts the normal developmental regulation of poison exon inclusion leads to reduced Nav1.1 and DS pathophysiology.

The SWI/SNF subunits BRG1 affects alternative splicing by changing RNA binding factor interactions with RNA

BRG1 and BRM are ATPase core subunits of the human SWI/SNF chromatin remodelling complexes. The function of the SWI/SNF complexes in transcriptional initiation has been well studied, while a function in alternative splicing has only been studied for a few cases for BRM-containing SWI/SNF complexes. Here, we have expressed BRG1 in C33A cells, a BRG1 and BRM-deficient cell line, and we have analysed the effects on the transcriptome by RNA sequencing. We have shown that BRG1 expression affects the splicing of a subset of genes. For some, BRG1 expression favours exon inclusion and for others, exon skipping. Some of the changes in alternative splicing induced by BRG1 expression do not require the ATPase activity of BRG1. Among the exons regulated through an ATPase-independent mechanism, the included exons had signatures of high GC-content and lacked a positioned nucleosome at the exon. By investigating three genes in which the expression of either wild-type BRG1 or a BRG1-ATPase-deficient variant favoured exon inclusion, we showed that expression of the ATPases promotes the local recruitment of RNA binding factors to chromatin and RNA in a differential manner. The hnRNPL, hnRNPU and SAM68 proteins associated to chromatin in C33A cells expressing BRG1 or BRM, but their association with RNA varied. We propose that SWI/SNF can regulate alternative splicing by interacting with splicing-RNA binding factor and altering their binding to the nascent pre-mRNA, which changes RNP structure.Author summarySplicing, in particular alternative splicing, is a combinatorial process which involves splicing factor complexes and many RNA binding splicing regulatory proteins in different constellations. Most splicing events occur during transcription, which also makes the DNA sequence, the chromatin state and the transcription rate at the exons important components that influence the splicing outcome. We show here that the ATP-dependent chromatin remodelling complex SWI/SNF influences the interactions of splicing regulatory factors with RNA during transcription on certain exons that have a high GC-content. The splicing on this type of exon rely on the ATPase BRG1 and favour inclusion of alternative exons in an ATP-independent manner. SWI/SNF complexes are known to alter the chromatin structure at promoters in transcription initiation, and have been previously shown to alter the transcription rate or nucleosome position in splicing. Our results suggests a further mechanism for chromatin remodelling proteins in splicing: to change the interaction patterns of RNA binding splicing regulatory factors at alternative exons to alter the splicing outcome.

Spliceosomal Disruption of the Non-Canonical SWI/SNF Chromatin Remodeling Complex in SF3B1 Mutant Leukemias

Mutations in the RNA splicing factor SF3B1 are common in MDS and other myeloid malignancies. SF3B1 mutations promote expression of mRNAs that use an aberrant, intron proximal 3' splice site (ss). Despite the consistency of this finding, linking aberrant splicing changes to disease pathogenesis has been a challenge. Here we identify aberrant splicing and downregulated expression of BRD9, a member of the recently described ATP-dependent non-canonical BAF (ncBAF) chromatin remodeling complex, across SF3B1 mutant leukemias. In so doing, we identify a novel role for altered ncBAF function in hematopoiesis and MDS. To systematically identify functionally important aberrant splicing events created by mutant SF3B1, we integrated differential splicing events in SF3B1 mutant versus wild-type MDS with a positive enrichment CRISPR screen mimicking splicing changes induced by mutant SF3B1 that promote NMD (non-sense mediated mRNA decay). We tested whether loss of any gene functionally inactivated by SF3B1 mutations promoted transformation of Ba/F3 and 32D cells. This identified a specific NMD-inducing aberrant splicing event in BRD9 which promoted cytokine independence (Fig. A) and exhibited striking aberrant splicing across CLL and MDS and across all mutational hotspots in SF3B1 (Fig. B). SF3B1 mutations cause exonization of a normally intronic sequence in BRD9, resulting in inclusion of a poison exon that interrupts BRD9's reading frame (Fig. C) and reduced BRD9 mRNA and protein expression through NMD (Fig. D). We confirmed that mutant SF3B1 suppressed full-length BRD9 levels without generating truncated BRD9 protein. Loss of BRD9 impaired ncBAF complex formation as indicated by abolished interaction between the ncBAF specific component GLTSCR1 and the ATPase subunit BRG1 upon chemical or spliceosomal BRD9 ablation (Fig. D). Given that prior work has linked mutant SF3B1 to use of aberrant 3' ss, we sought to understand the molecular basis for aberrant exon inclusion in BRD9 by mutant SF3B1. Lariat sequencing of SF3B1 mutant versus WT K562 cells and BRD9 minigene analyses identified use of a deep intronic branchpoint adenosine by mutant SF3B1 to promote BRD9 poison exon inclusion (Fig. E). The data above suggest a role for BRD9 downregulation in SF3B1 mutant leukemia. While prior work indicated that BRD9 is required in MLL-rearranged AML (Hohmman et al. Nature Chemical Biology 2016), the role of BRD9 in normal hematopoiesis or other subtypes of myeloid neoplasms has not been evaluated. Genetic downregulation of BRD9 in normal human hematopoietic progenitors from cord blood promoted myelopoiesis while impairing megakaryopoiesis. Interestingly and unexpectedly, BRD9 loss in CD34+ cells promoted terminal erythroid differentiation in vitro. To further evaluate BRD9's role in hematopoiesis in vivo, we also generated mice with inducible knockout of the bromodomain of BRD9 (required for BRD9 function) and generation of a frameshift transcript resulting in reduced Brd9 expression (Fig. F). Loss of Brd9 resulted in macrocytosis with bone marrow erythroid dysplasia in a dosage-dependent manner, along with impaired lymphopoiesis and myeloid skewing. Moreover, competitive transplantation of hematopoietic precursors from these mice revealed that ablation of Brd9 function impaired lymphoid reconstitution while promoting advantage of myeloid cells and hematopoietic precursors (Fig. G-I). In myeloid leukemia cells, introduction of SF3B1K700E or downregulation of BRD9 resulted in increased chromatin accessibility at promoters with a significant overlap in commonly upregulated genes. This finding suggests shared epigenetic effects of SF3B1K700E mutations and BRD9 loss (Fig. J). These data identify aberrant splicing of BRD9 across the spectrum of SF3B1 mutant cancers and identify a novel role for downregulation of ncBAF function in MDS pathogenesis. Consistent with human genetic data, genetic ablation of BRD9 function in mouse and human hematopoietic cells resulted in myeloid skewing and dyserythropoiesis. These data suggest that targeted correction of aberrant BRD9 splicing might serve as a novel therapeutic approach for SF3B1-mutant leukemias. Of note, treatment with drugs impairing the binding of mutant SF3B1 to RNA resulted in a dose-dependent rescue of aberrant BRD9 splicing in vitro (Fig. K) and in treatment of an SF3B1 mutant AML patient-derived xenograft in vivo. Figure Disclosures Kadoch: Foghorn Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.