GPATCH8 modulates mutant SF3B1 mis-splicing and pathogenicity in hematologic malignancies.

Mutations in the RNA splicing factor gene SF3B1 are common across hematologic and solid cancers and result in widespread alterations in splicing, yet there is currently no therapeutic means to correct this mis-splicing. Here, we utilize synthetic introns uniquely responsive to mutant SF3B1 to identify trans factors required for aberrant mutant SF3B1 splicing activity. This revealed the G-patch domain-containing protein GPATCH8 as required for mutant SF3B1-induced splicing alterations and impaired hematopoiesis. GPATCH8 is involved in quality control of branchpoint selection, interacts with the RNA helicase DHX15, and functionally opposes SURP and G-patch domain containing 1 (SUGP1), a G-patch protein recently implicated in SF3B1-mutant diseases. Silencing of GPATCH8 corrected one-third of mutant SF3B1-dependent splicing defects and was sufficient to improve dysfunctional hematopoiesis in SF3B1-mutant mice and primary human progenitors. These data identify GPATCH8 as a novel splicing factor required for mis-splicing by mutant SF3B1 and highlight the therapeutic impact of correcting aberrant splicing in SF3B1-mutant cancers.

Molecular impact of mutations in RNA splicing factors in cancer.

Somatic mutations in genes encoding components of the RNA splicing machinery occur frequently in multiple forms of cancer. The most frequently mutated RNA splicing factors in cancer impact intronic branch site and 3' splice site recognition. These include mutations in the core RNA splicing factor SF3B1 as well as mutations in the U2AF1/2 heterodimeric complex, which recruits the SF3b complex to the 3' splice site. Additionally, mutations in splicing regulatory proteins SRSF2 and RBM10 are frequent in cancer, and there has been a recent suggestion that variant forms of small nuclear RNAs (snRNAs) may contribute to splicing dysregulation in cancer. Here, we describe molecular mechanisms by which mutations in these factors alter splice site recognition and how studies of this process have yielded new insights into cancer pathogenesis and the molecular regulation of splicing. We also discuss data linking mutant RNA splicing factors to RNA metabolism beyond splicing.

Transcription elongation defects link oncogenic SF3B1 mutations to targetable alterations in chromatin landscape.

Transcription and splicing of pre-messenger RNA are closely coordinated, but how this functional coupling is disrupted in human diseases remains unexplored. Using isogenic cell lines, patient samples, and a mutant mouse model, we investigated how cancer-associated mutations in SF3B1 alter transcription. We found that these mutations reduce the elongation rate of RNA polymerase II (RNAPII) along gene bodies and its density at promoters. The elongation defect results from disrupted pre-spliceosome assembly due to impaired protein-protein interactions of mutant SF3B1. The decreased promoter-proximal RNAPII density reduces both chromatin accessibility and H3K4me3 marks at promoters. Through an unbiased screen, we identified epigenetic factors in the Sin3/HDAC/H3K4me pathway, which, when modulated, reverse both transcription and chromatin changes. Our findings reveal how splicing factor mutant states behave functionally as epigenetic disorders through impaired transcription-related changes to the chromatin landscape. We also present a rationale for targeting the Sin3/HDAC complex as a therapeutic strategy.

m6A-driven SF3B1 translation control steers splicing to direct genome integrity and leukemogenesis.

SF3B1 is the most mutated splicing factor (SF) in myelodysplastic syndromes (MDSs), which are clonal hematopoietic disorders with variable risk of leukemic transformation. Although tumorigenic SF3B1 mutations have been extensively characterized, the role of "non-mutated" wild-type SF3B1 in cancer remains largely unresolved. Here, we identify a conserved epitranscriptomic program that steers SF3B1 levels to counteract leukemogenesis. Our analysis of human and murine pre-leukemic MDS cells reveals dynamic regulation of SF3B1 protein abundance, which affects MDS-to-leukemia progression in vivo. Mechanistically, ALKBH5-driven 5' UTR m6A demethylation fine-tunes SF3B1 translation directing splicing of central DNA repair and epigenetic regulators during transformation. This impacts genome stability and leukemia progression in vivo, supporting an integrative analysis in humans that SF3B1 molecular signatures may predict mutational variability and poor prognosis. These findings highlight a post-transcriptional gene expression nexus that unveils unanticipated SF3B1-dependent cancer vulnerabilities.

Efficient RNA polymerase II pause release requires U2 snRNP function.

Transcription by RNA polymerase II (Pol II) is coupled to pre-mRNA splicing, but the underlying mechanisms remain poorly understood. Co-transcriptional splicing requires assembly of a functional spliceosome on nascent pre-mRNA, but whether and how this influences Pol II transcription remains unclear. Here we show that inhibition of pre-mRNA branch site recognition by the spliceosome component U2 snRNP leads to a widespread and strong decrease in new RNA synthesis from human genes. Multiomics analysis reveals that inhibition of U2 snRNP function increases the duration of Pol II pausing in the promoter-proximal region, impairs recruitment of the pause release factor P-TEFb, and reduces Pol II elongation velocity at the beginning of genes. Our results indicate that efficient release of paused Pol II into active transcription elongation requires the formation of functional spliceosomes and that eukaryotic mRNA biogenesis relies on positive feedback from the splicing machinery to the transcription machinery.

(G)Patching up mis-splicing in cancer.

Benbarche, Pineda, Galvis, et al. delineate an essential role for the G-patch motif-containing protein GPATCH8 in mis-splicing associated with cancer-driving mutations of the splicing factor SF3B1. GPATCH8 cooperates with SF3B1 mutants, affecting the splicing machinery. Targeting GPATCH8 reveals therapeutic opportunities for SF3B1 mutant cancers and other splicing-related diseases.

Selective inhibition of CDK7 reveals high-confidence targets and new models for TFIIH function in transcription.

CDK7 associates with the 10-subunit TFIIH complex and regulates transcription by phosphorylating the C-terminal domain (CTD) of RNA polymerase II (RNAPII). Few additional CDK7 substrates are known. Here, using the covalent inhibitor SY-351 and quantitative phosphoproteomics, we identified CDK7 kinase substrates in human cells. Among hundreds of high-confidence targets, the vast majority are unique to CDK7 (i.e., distinct from other transcription-associated kinases), with a subset that suggest novel cellular functions. Transcription-associated factors were predominant CDK7 substrates, including SF3B1, U2AF2, and other splicing components. Accordingly, widespread and diverse splicing defects, such as alternative exon inclusion and intron retention, were characterized in CDK7-inhibited cells. Combined with biochemical assays, we establish that CDK7 directly activates other transcription-associated kinases CDK9, CDK12, and CDK13, invoking a "master regulator" role in transcription. We further demonstrate that TFIIH restricts CDK7 kinase function to the RNAPII CTD, whereas other substrates (e.g., SPT5 and SF3B1) are phosphorylated by the three-subunit CDK-activating kinase (CAK; CCNH, MAT1, and CDK7). These results suggest new models for CDK7 function in transcription and implicate CAK dissociation from TFIIH as essential for kinase activation. This straightforward regulatory strategy ensures CDK7 activation is spatially and temporally linked to transcription, and may apply toward other transcription-associated kinases.

Etiology of craniofacial and cardiac malformations in a mouse model of SF3B4-related syndromes.

Pathogenic variants in SF3B4, a component of the U2 snRNP complex important for branchpoint sequence recognition and splicing, are responsible for the acrofacial disorders Nager and Rodriguez Syndrome, also known as SF3B4-related syndromes. Patients exhibit malformations in the head, face, limbs, vertebrae as well as the heart. To uncover the etiology of craniofacial malformations found in SF3B4-related syndromes, mutant mouse lines with homozygous deletion of Sf3b4 in neural crest cells (NCC) were generated. Like in human patients, these embryos had craniofacial and cardiac malformations with variable expressivity and penetrance. The severity and survival of Sf3b4 NCC mutants was modified by the level of Sf3b4 in neighboring non-NCC. RNA sequencing analysis of heads of embryos prior to morphological abnormalities revealed significant changes in expression of genes forming the NCC regulatory network, as well as an increase in exon skipping. Additionally, several key histone modifiers involved in craniofacial and cardiac development showed increased exon skipping. Increased exon skipping was also associated with use of a more proximal branch point, as well as an enrichment in thymidine bases in the 50 bp around the branch points. We propose that decrease in Sf3b4 causes changes in the expression and splicing of transcripts required for proper craniofacial and cardiac development, leading to abnormalities.

Broad variation in response of individual introns to splicing inhibitors in a humanized yeast strain.

Intron branchpoint (BP) recognition by the U2 snRNP is a critical step of splicing, vulnerable to recurrent cancer mutations and bacterial natural product inhibitors. The BP binds a conserved pocket in the SF3B1 (human) or Hsh155 (yeast) U2 snRNP protein. Amino acids that line this pocket affect the binding of splicing inhibitors like Pladienolide-B (Plad-B), such that organisms differ in their sensitivity. To study the mechanism of splicing inhibitor action in a simplified system, we modified the naturally Plad-B resistant yeast Saccharomyces cerevisiae by changing 14 amino acids in the Hsh155 BP pocket to those from human. This humanized yeast grows normally, and splicing is largely unaffected by the mutation. Splicing is inhibited within minutes after the addition of Plad-B, and different introns appear inhibited to different extents. Intron-specific inhibition differences are also observed during cotranscriptional splicing in Plad-B using single-molecule intron tracking to minimize gene-specific transcription and decay rates that cloud estimates of inhibition by standard RNA-seq. Comparison of Plad-B intron sensitivities to those of the structurally distinct inhibitor Thailanstatin-A reveals intron-specific differences in sensitivity to different compounds. This work exposes a complex relationship between the binding of different members of this class of inhibitors to the spliceosome and intron-specific rates of BP recognition and catalysis. Introns with variant BP sequences seem particularly sensitive, echoing observations from mammalian cells, where monitoring individual introns is complicated by multi-intron gene architecture and alternative splicing. The compact yeast system may hasten the characterization of splicing inhibitors, accelerating improvements in selectivity and therapeutic efficacy.

Study of the RNA splicing kinetics via in vivo 5-EU labeling.

Splicing is an important step of gene expression in all eukaryotes. Splice sites might be used with different efficiency, giving rise to alternative splicing products. At the same time, splice sites might be used at a variable rate. We used 5-ethynyl uridine labeling to sequence a nascent transcriptome of HeLa cells and deduced the rate of splicing for each donor and acceptor splice site. The following correlation analysis showed a correspondence of primary transcript features with the rate of splicing. Some dependencies we revealed were anticipated, such as a splicing rate decrease with a decreased complementarity of the donor splice site to U1 and acceptor sites to U2 snRNAs. Other dependencies were more surprising, like a negative influence of a distance to the 5' end on the rate of the acceptor splicing site utilization, or the differences in splicing rate between long, short, and RBM17-dependent introns. We also observed a deceleration of last intron splicing with an increase of the distance to the poly(A) site, which might be explained by the cooperativity of the splicing and polyadenylation. Additional analysis of splicing kinetics of SF3B4 knockdown cells suggested the impairment of a U2 snRNA recognition step. As a result, we deconvoluted the effects of several examined features on the splicing rate into a single regression model. The data obtained here are useful for further studies in the field, as they provide general splicing rate dependencies as well as help to justify the existence of slowly removed splice sites.

Structural Basis of Splicing Modulation by Antitumor Macrolide Compounds.

SF3B is a multi-protein complex essential for branch site (BS) recognition and selection during pre-mRNA splicing. Several splicing modulators with antitumor activity bind SF3B and thereby modulate splicing. Here we report the crystal structure of a human SF3B core in complex with pladienolide B (PB), a macrocyclic splicing modulator and potent inhibitor of tumor cell proliferation. PB stalls SF3B in an open conformation by acting like a wedge within a hinge, modulating SF3B's transition to the closed conformation needed to form the BS adenosine-binding pocket and stably accommodate the BS/U2 duplex. This work explains the structural basis for the splicing modulation activity of PB and related compounds, and reveals key interactions between SF3B and a common pharmacophore, providing a framework for future structure-based drug design.

The Output of Protein-Coding Genes Shifts to Circular RNAs When the Pre-mRNA Processing Machinery Is Limiting.

Many eukaryotic genes generate linear mRNAs and circular RNAs, but it is largely unknown how the ratio of linear to circular RNA is controlled or modulated. Using RNAi screening in Drosophila cells, we identify many core spliceosome and transcription termination factors that control the RNA outputs of reporter and endogenous genes. When spliceosome components were depleted or inhibited pharmacologically, the steady-state levels of circular RNAs increased while expression of their associated linear mRNAs concomitantly decreased. Upon inhibiting RNA polymerase II termination via depletion of the cleavage/polyadenylation machinery, circular RNA levels were similarly increased. This is because readthrough transcripts now extend into downstream genes and are subjected to backsplicing. In total, these results demonstrate that inhibition or slowing of canonical pre-mRNA processing events shifts the steady-state output of protein-coding genes toward circular RNAs. This is in part because nascent RNAs become directed into alternative pathways that lead to circular RNA production.

TP53 variant allele frequency and therapy-related setting independently predict survival in myelodysplastic syndromes with del(5q).

Among 210 patients with myelodysplastic syndromes (MDSs) with del(5q), molecular information was available at diagnosis or at least 3 months before leukaemic transformation in 146 cases. Multivariate analysis identified therapy-related setting (p = 0.02; HR 2.3) and TP53 variant allele frequency (VAF) ≥22% (p < 0.01; HR 2.8), but not SF3B1 mutation (p = 0.65), as independent risk factors for survival. Median survival was 11.7 versus 4 years (5/10-year survival 73%/52% vs. 42%/14%) in the absence (N = 112) versus presence (N = 34) of ≥1 risk factors; leukaemia-free survival was affected by TP53 VAF ≥22% (p < 0.01). Such information might inform treatment decision-making in MDS-del(5q) regarding allogeneic stem cell transplant.

Validation and comparison of 5th edition World Health Organization classification (WHO 2022) and International Consensus Classification proposals for MDS-SFB31.

The criteria of myelodysplastic syndromes (MDS) with mutated SFB31 (MDS-SFB31) proposed by the 5th edition of the WHO classification (WHO 2022) and the International Consensus Classification (ICC) need validation. We analysed 125 consecutive MDS cases with SFB31 mutation or ring sideroblasts (RS) ≥15% without excess blasts. We found that SFB31-negative MDS with RS had significantly different clinical features and worse prognosis. According to WHO 2022, the detection of ≥15% RS may substitute for SF3B1 mutation and our analyses support this proposal for similar prognosis of two groups after excluding high-risk genetic features referred by WHO 2022. Patients with variant allele frequency (VAF) <10% SFB31 tend to have briefer survival, supporting the VAF 10% threshold of ICC. Patients with multilineage dysplasia (MLD) had significantly shorter OS than those with single lineage dysplasia. MLD is still a powerful morphological marker of worse outcome in WHO 2022 and ICC-defined MDS-SF3B1.

SF3B2 Haploinsufficiency Associated With Hirschprung Disease and Complex Cardiac Defect Without Craniofacial Microsomia.

Haploinsufficiency of SF3B2 is associated with craniofacial microsomia, characterized by mandibular hypoplasia and microtia, often with preauricular tags or pits, epibulbar dermoids, and cleft palate. In addition, extracraniofacial anomalies may be present, such as skeletal, cardiac renal, and abnormalities of the central nervous system. Variants have been either de novo or inherited, and both inter- and intrafamilial variability has been observed. Here we describe a patient referred for exome sequencing for a complex congenital heart defect and Hirschsprung disease found by exome sequencing to be heterozygous for a loss of function variant, c.945dup (p.Val316SerfsTer5), in SF3B2. This variant was inherited from a parent with an isolated cardiac defect. Interestingly, neither have the defining craniofacial features or other dysmorphisms. This report further illustrates the degree of phenotypic variability seen in SF3B2-related disease and expands the spectrum to include Hirschsprung disease.

Prognostic impact of DTA mutation and co-occurring mutations in patients with myelodysplastic syndrome.

OBJECTIVE: To evaluate the frequency and prognostic significance of DTA (DNMT3A、TET2、ASXL1) gene mutation and co-occurring mutations in patients with myelodysplastic syndrome (MDS). METHODS: The clinical data of 102 newly diagnosed MDS patients who accepted Next Generation Sequencing (NGS) was retrospectively analyzed. According to whether the patients had DTA gene mutation, the patients were divided into DTA mutated (DTA-mut) group and wild type (DTA-wt) group, and the relationship between gene mutation and clinical characteristics and prognosis was analyzed. RESULTS: Among the 102 MDS patients, 96% (98/102) presented with mutation, while the mean number of mutations was 3.04 mutations/patient. DTA-mut was detected in 56.9% (58/102) patients. The most frequent co-mutated genes in DTA-mut group were SF3B1 (25.8%), RUNX1 (24.1%), U2AF1 (18.9%), SRSF2, EZH2, SETBP1 (17.2%), STAG2 (15.5%), IDH2 (12.1%) and BCOR, CBL (10.3%). The two groups showed no significant differences in ages, blood parameters, bone marrow blasts, WHO 2022 classification, IPSS-R risk category and rate of conversion to leukemia. Compared with the DTA-wt group, the mutation frequency of RUNX1 was higher (P = 0.02), while mutation frequency of TP53 was lower (P = 0.001) and the mutation frequency of ≥ 3 co-mutated genes was higher in DTA-mut group (P = 0.00). Survival analysis showed that the overall survivals (OS) of DTA-mut patients was significantly inferior to that of DTA-wt patients (P = 0.0332). According to IPSS-R classification, a statistically significant difference in OS was only observed in higher risk (IPSS-R > 3.5) group (P = 0.0058). In the context of DTA mutation, the OS of patients with RUNX1 mutation was shorter than that of patients without RUNX1 mutation significantly (P = 0.0074). The OS of patients with SF3B1 mutation was longer than that of patients without SF3B1 mutation, but there was no statistical difference (P = 0.0827). DTA mutations were not independent prognostic factors when DTA and co-mutated genes with frequency > 10% were considered in Cox regression model (P = 0.329). However, multivariate analysis confirmed an independently adverse prognosis of RUNX1 co-mutation (P = 0.042, HR = 2.426, 95% CI:1.031-5.711) in DTA-mut cohort. Moreover, our multivariable analysis suggests that SRSF2-mut was an independent poor prognostic factor for all MDS patients (P = 0.047), but lost significance (P = 0.103) for DTA-mut patients. CONCLUSIONS: DTA mutations are frequently observed in patients with MDS, often accompanied by genes involved in RNA splicing and transcription factors like SF3B1 and RUNX1. DTA and concomitant mutations affect prognosis in MDS patients and RUNX1 was identified as an independent poor prognostic factor in patients with DTA mutations.

Molecular Architecture of SF3b and Structural Consequences of Its Cancer-Related Mutations.

SF3b is a heptameric protein complex of the U2 small nuclear ribonucleoprotein (snRNP) that is essential for pre-mRNA splicing. Mutations in the largest SF3b subunit, SF3B1/SF3b155, are linked to cancer and lead to alternative branch site (BS) selection. Here we report the crystal structure of a human SF3b core complex, revealing how the distinctive conformation of SF3b155's HEAT domain is maintained by multiple contacts with SF3b130, SF3b10, and SF3b14b. Protein-protein crosslinking enabled the localization of the BS-binding proteins p14 and U2AF65 within SF3b155's HEAT-repeat superhelix, which together with SF3b14b forms a composite RNA-binding platform. SF3b155 residues, the mutation of which leads to cancer, contribute to the tertiary structure of the HEAT superhelix and its surface properties in the proximity of p14 and U2AF65. The molecular architecture of SF3b reveals the spatial organization of cancer-related SF3b155 mutations and advances our understanding of their effects on SF3b structure and function.

Sf3b4 regulates chromatin remodeler splicing and Hox expression.

SF3B proteins form a heptameric complex in the U2 small nuclear ribonucleoprotein, essential for pre-mRNA splicing. Heterozygous pathogenic variants in human SF3B4 are associated with head, face, limb, and vertebrae defects. Using the CRISPR/Cas9 system, we generated mice with constitutive heterozygous deletion of Sf3b4 and showed that mutant embryos have abnormal vertebral development. Vertebrae abnormalities were accompanied by changes in levels and expression pattern of Hox genes in the somites. RNA sequencing analysis of whole embryos and somites of Sf3b4 mutant and control litter mates revealed increased expression of other Sf3b4 genes. However, the mutants exhibited few differentially expressed genes and a large number of transcripts with differential splicing events (DSE), predominantly increased exon skipping and intron retention. Transcripts with increased DSE included several genes involved in chromatin remodeling that are known to regulate Hox expression. Our study confirms that Sf3b4 is required for normal vertebrae development and shows, for the first time, that like Sf3b1, Sf3b4 also regulates Hox expression. We propose that abnormal splicing of chromatin remodelers is primarily responsible for vertebral defects found in Sf3b4 heterozygous mutant embryos.

Spliceosomal gene mutations in myelodysplasia: molecular links to clonal abnormalities of hematopoiesis.

Genomic analyses of the myeloid malignancies and clonal disorders of hematopoiesis that may give rise to these disorders have identified that mutations in genes encoding core spliceosomal proteins and accessory regulatory splicing factors are among the most common targets of somatic mutations. These spliceosomal mutations often occur in a mutually exclusive manner with one another and, in aggregate, account for the most frequent class of mutations in patients with myelodysplastic syndromes (MDSs) in particular. Although substantial progress has been made in understanding the effects of several of these mutations on splicing and splice site recognition, functional connections linking the mechanistic changes in splicing induced by these mutations to the phenotypic consequences of clonal and aberrant hematopoiesis are not yet well defined. This review describes our current understanding of the mechanistic and biological effects of spliceosomal gene mutations in MDSs as well as the regulation of splicing throughout normal hematopoiesis.

SF3B1/Hsh155 HEAT motif mutations affect interaction with the spliceosomal ATPase Prp5, resulting in altered branch site selectivity in pre-mRNA splicing.

Mutations in the U2 snRNP component SF3B1 are prominent in myelodysplastic syndromes (MDSs) and other cancers and have been shown recently to alter branch site (BS) or 3' splice site selection in splicing. However, the molecular mechanism of altered splicing is not known. We show here that hsh155 mutant alleles in Saccharomyces cerevisiae, counterparts of SF3B1 mutations frequently found in cancers, specifically change splicing of suboptimal BS pre-mRNA substrates. We found that Hsh155p interacts directly with Prp5p, the first ATPase that acts during spliceosome assembly, and localized the interacting regions to HEAT (Huntingtin, EF3, PP2A, and TOR1) motifs in SF3B1 associated with disease mutations. Furthermore, we show that mutations in these motifs from both human disease and yeast genetic screens alter the physical interaction with Prp5p, alter branch region specification, and phenocopy mutations in Prp5p. These and other data demonstrate that mutations in Hsh155p and Prp5p alter splicing because they change the direct physical interaction between Hsh155p and Prp5p. This altered physical interaction results in altered loading (i.e., "fidelity") of the BS-U2 duplex into the SF3B complex during prespliceosome formation. These results provide a mechanistic framework to explain the consequences of intron recognition and splicing of SF3B1 mutations found in disease.