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Alternative splicing events associated with spliceosome mutations. a, b, c Scatter plots comparing mean PSI values between the SF3B1- (a), SRSF2- (b), and U2AF1-mutated samples (c), and those without SF mutations. The number of samples in each group is shown in Fig. 1c. Significantly associated events (q-value <0.01) are color coded as in the bottom right panel. The panels in the right are scatter plots for each splicing pattern, also showing the number of significant events. Other types of alternative exon usage included mutually exclusive exons, alternative first exon, and alternative last exon
Source publication
Spliceosome mutations are frequently found in myelodysplasia. Splicing alterations induced by these mutations, their precise targets, and the effect at the transcript level have not been fully elucidated. Here we report transcriptomic analyses of 265 bone marrow samples from myelodysplasia patients, followed by a validation using CRISPR/Cas9-mediat...
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Here we show that the serine/arginine rich splicing factor 2 (SRSF2) promotes cryptic 3′ splice-site (3′AG′) usage during cassette exon exclusion in survival of motor neuron (SMN2) minigenes. Deletion of the 3′AG′ (3′AG′1), its associated branch point (BP′) and polypyrimidine tract (PPT′) sequences directs SRSF2 to promote a second 3′AG′ (3′AG′2) w...
Altered splicing contributes to the pathogenesis of human blood disorders including myelodysplastic syndromes (MDS) and leukemias. Here we characterize the transcriptomic regulation of PRPF40B, which is a splicing factor mutated in a small fraction of MDS patients. We generated a full PRPF40B knockout (KO) in the K562 cell line by CRISPR/Cas9 techn...
Citations
... Mutations in components of the splicing machinery as a cause of aberrant AS have been found in four genes among those listed in Supplementary Table S1, namely EZH2, GNAS Complex Locus (GNAS), Patched 1 (PTCH1), and Cyclin D2 (CCND2). In the two former cases, mutations in both U2AF1 and SRSF2 result in aberrant AS [111][112][113]. The splicing of EZH2 pre-mRNA exhibits multiple other features that will be dealt with below. ...
Cancer driver genes are either oncogenes or tumour suppressor genes that are classically activated or inactivated, respectively, by driver mutations. Alternative splicing-which produces various mature mRNAs and, eventually, protein variants from a single gene-may also result in driving neoplastic transformation because of the different and often opposed functions of the variants of driver genes. The present review analyses the different alternative splicing events that result in driving neoplastic transformation, with an emphasis on their molecular mechanisms. To do this, we collected a list of 568 gene drivers of cancer and revised the literature to select those involved in the alternative splicing of other genes as well as those in which its pre-mRNA is subject to alternative splicing, with the result, in both cases, of producing an oncogenic isoform. Thirty-one genes fall into the first category, which includes splicing factors and components of the spliceosome and splicing regulators. In the second category, namely that comprising driver genes in which alternative splicing produces the oncogenic isoform, 168 genes were found. Then, we grouped them according to the molecular mechanisms responsible for alternative splicing yielding oncogenic isoforms, namely, mutations in cis splicing-determining elements, other causes involving non-mutated cis elements, changes in splicing factors, and epigenetic and chromatin-related changes. The data given in the present review substantiate the idea that aberrant splicing may regulate the activation of proto-oncogenes or inactivation of tumour suppressor genes and details on the mechanisms involved are given for more than 40 driver genes.
... It is important to note, that SF3B1 mutations alter splicing patterns with numerous AS events. Despite the high incidence of 3′ splice site alterations in SF3B1 mutant cells, a small but global reduction of intronretaining variants was reported to be the most frequent aberrant splicing event in MDS samples with SF3B1 mutation (Shiozawa et al. 2018). Although the functional relevance of the SF3B1 mutation-induced splicing events in tumor cells is not well defined, their effects might serve as a source of neoantigens for the development of personalized vaccines or adoptive cell-based therapies (Schischlik et al. 2019). ...
Alternative splicing (AS) is a strictly regulated process that generates multiple mRNA variants from a single gene, thus contributing to proteome diversity. Transcriptome-wide sequencing studies revealed networks of functionally coordinated splicing events, which produce isoforms with distinct or even opposing functions. To date, several mechanisms of AS are deregulated in leukemic cells, mainly due to mutations in splicing and/or epigenetic regulators and altered expression of splicing factors (SFs). In this review, we discuss aberrant splicing events induced by mutations affecting SFs ( SF3B1 , U2AF1 , SRSR2 , and ZRSR2 ), spliceosome components ( PRPF8 , LUC7L2 , DDX41, and HNRNPH1 ), and epigenetic modulators ( IDH1 and IDH2 ). Finally, we provide an extensive overview of the biological relevance of aberrant isoforms of genes involved in the regulation of apoptosis (e. g. BCL-X , MCL-1 , FAS , and c-FLIP ), activation of key cellular signaling pathways ( CASP8 , MAP3K7 , and NOTCH2 ), and cell metabolism ( PKM ).
... Mutations in splicing factors have been implicated in the pathogenesis of MDS and AML, most likely via abnormal RNA splicing, with exon skipping events, rather than loss of function [16,17]. Moreover, SRSF2 and SF3B1 mutations have been associated with in vitro resistance to VEN in primary AML samples [18]. ...
... For example, ASXL1 mutations confer VEN sensitivity in preclinical models but not in clinical studies [57,58]. Mutations in splicing factors, including SRSF2, U2AF1, SF3B1, and ZRSR2, have been implicated in the pathogenesis of MDS and AML [59,60]. These mutations are encountered in approximately 50% of secondary AML cases and usually correlate with inferior outcomes to ICT [49,61,62]. ...
Simple Summary
Venetoclax–azacitidine is a new standard for elderly or unfit acute myeloid leukemia patients. Nevertheless, resistance remains a matter of concern. The main genetic alterations associated with venetoclax sensitivity are IDH mutations, whereas TP53, signaling mutations, and BAX mutations are associated with venetoclax resistance. Non-genetic resistance mechanisms have also been described, including changes in apoptotic proteins, differentiation states, metabolic status, and mitochondrial machinery. Venetoclax-based triplet therapies including IDH and FLT3 inhibitors or innovative therapies are under investigation to target resistances.
Abstract
Venetoclax is a BH3-mimetics agent interacting with the anti-apoptotic protein BCL2, facilitating cytochrome c release from mitochondria, subsequent caspases activation, and cell death. Venetoclax combined with azacitidine (VEN-AZA) has become a new standard treatment for AML patients unfit for intensive chemotherapy. In the phase III VIALE-A study, VEN-AZA showed a 65% overall response rate and 14.7 months overall survival in comparison with 22% and 8 months in the azacitidine monotherapy control arm. Despite these promising results, relapses and primary resistance to venetoclax are frequent and remain an unmet clinical need. Clinical and preclinical studies have been conducted to identify factors driving resistance. Among them, the most documented are molecular alterations including IDH, FLT3, TP53, and the newly described BAX mutations. Several non-genetic factors are also described such as metabolic plasticity, changes in anti-apoptotic protein expression, and dependencies, as well as monocytic differentiation status. Strategies to overcome venetoclax resistance are being developed in clinical trials, including triplet therapies with targeted agents targeting IDH, FLT3, as well as the recently developed menin inhibitors or immunotherapies such as antibody–drug conjugated or monoclonal antibodies. A better understanding of the molecular factors driving venetoclax resistance by single-cell analyses will help the discovery of new therapeutic strategies in the future.
... Splicing is a fundamental step in eukaryotic gene expression in which non-coding introns are removed from pre-messenger RNA (pre-mRNA) transcripts and exons are joined to form mature mRNAs. This intricate process is often disrupted in cancer, either by mutations in spliceosomal genes or by other mechanisms that affect normal splicing function (1)(2)(3)(4)(5). In turn, this can lead to changes in the composition of expressed isoforms and the formation of new isoforms that alter the encoded proteins and can have far-reaching consequences for cellular function. ...
... Previous studies based on short-read RNA sequencing (RNA-seq) have reported alternative 3' splice site usage (3'AS) and intron retention (IR) as the most prominent splicing alterations in CLL and MDS patients with mutated SF3B1 (3,(15)(16)(17)(18). The alternative 3' splice sites (referred to as AG') that were preferably used upon SF3B1 mutation, are enriched at approximately 20 nucleotides (nt) upstream of the canonical splice sites (AG) (15)(16)(17)19). ...
... In total, we identified 775 differential splicing events in 530 different genes (Fig. 3a, Supplementary Table S3). As reported previously (3,(15)(16)(17)(18)24), the splicing changes upon SF3B1 mutation were strongly enriched for 3'AS (326, 42%) and IR (213, 27%) events which together accounted for more than two thirds of the significant changes. The majority of IR events showed decreased IR (89%), whereas the majority of 3'AS events (78%) showed higher PSI values, corresponding to longer exons in SF3B1 mut/wt (Fig. 3b,c, Supplementary Fig. S6, Supplementary Table S3). ...
Background
Mutations in splicing factor 3B subunit 1 ( SF3B1 ) frequently occur in patients with chronic lymphocytic leukemia (CLL) and myelodysplastic syndromes (MDS). These mutations have a different effect on the disease prognosis with beneficial effect in MDS and worse prognosis in CLL patients. A full-length transcriptome approach can expand our knowledge on SF3B1 mutation effects on RNA splicing and its contribution to patient survival and treatment options.
Results
We applied long-read transcriptome sequencing to 44 MDS and CLL patients with and without SF3B1 mutations and found > 60% of novel isoforms. Splicing alterations were largely shared between cancer types and specifically affected the usage of introns and 3’ splice sites. Our data highlighted a constrained window at canonical 3’ splice sites in which dynamic splice site switches occurred in SF3B1 -mutated patients. Using transcriptome-wide RNA binding maps and molecular dynamics simulations, we showed multimodal SF3B1 binding at 3’ splice sites and predicted reduced RNA binding at the second binding pocket of SF3B1 K700E .
Conclusions
Our work presents the hitherto most complete long-read transcriptome sequencing study in CLL and MDS and provides a resource to study aberrant splicing in cancer. Moreover, we showed that different disease prognosis results most likely from the different cell types expanded during cancerogenesis rather than different mechanism of action of the mutated SF3B1 . These results have important implications for understanding the role of SF3B1 mutations in hematological malignancies and other related diseases.
Highlights
Long-read transcriptome sequencing data enables the identification of > 60% of novel isoforms in the transcriptomes of CLL and MDS patients and isogenic cell lines.
SF3B1 mutations trigger common splicing alterations upon SF3B1 mutations across patient cohorts, most frequently decreased intron retention and increased alternative 3’ splice site usage.
Mutation effect depends on alternative 3’ splice site and branch point positioning that coincide with bimodal SF3B1 binding at these sites
Molecular dynamics simulations predict reduced binding of SF3B1 K700E to mRNA at the second binding pocket harboring the polypyrimidine tract.
... SF3B1 mutations result in the use of cryptic 3' splice site upstream of the 3' splice site used in wild-type cells resulting in splicing changes [42,43]. Surprisingly, a low level but widespread reduction of intron retention isoforms (that is, enhanced splicing of regulated introns) seems to be the most frequent splicing alteration detected in bone marrow samples of SF3B1-mutated MDS [44]. ...
The process of alternative splicing (AS) is widely deregulated in a variety of cancers. Splicing is dependent upon splicing factors. Recently, several long noncoding RNAs (lncRNAs) have been shown to regulate AS by directly/indirectly interacting with splicing factors. This review focuses on the regulation of AS by lncRNAs through their interaction with splicing factors. AS mis-regulation caused by either mutation in splicing factors or deregulated expression of splicing factors and lncRNAs has been shown to be involved in cancer development and progression, making aberrant splicing, splicing factors and lncRNA suitable targets for cancer therapy. This review also addresses some of the current approaches used to target AS, splicing factors and lncRNAs. Finally, we discuss research challenges, some of the unanswered questions in the field and provide recommendations to advance understanding of the nexus of lncRNAs, AS and splicing factors in cancer.
... Alternative usage of different gene architectures enables to differential expression of various mRNA isoforms, including alternative transcription start/end sites and alternative splicing (AS) events, such as exon skipping, intron retention, alternative 5' and 3' splice sites [1][2][3] . The advances of single-cell transcriptomic technologies have provided a powerful tool to detect cellular heterogeneity in gene-level expression by using the sum of all transcripts originating from the same gene 4 . ...
... We developed a stepwise computational method CamoTSS to detect alternative transcription start site clusters and quantify their differential usage utilizing 5' tag-based scRNA-seq data alone (Fig. 1B). In brief, CamoTSS has three main steps after fetching TSS reads for a certain gene from an aligned bam file: (1) clustering of TSS reads with hierarchical clustering (minimum linkage distance: 100 bp by default), (2) filtering TSS clusters by technical thresholds (minimum UMIs: 50; minimum inter-cluster distance: 300 bp) and an embedded classifier to prevent TSS clusters from artefacts by using predictive features (see next paragraph) and (3) annotating these de-novo TSSs (by its summit position) to known annotations (e.g., GENCODE) optimized by a Hungarian algorithm while if a detected TSS cluster does not cover the optimal known TSS position, it remains called as new-TC or novel-TSS ( Fig. 1B; Methods). ...
Five-prime single-cell RNA-seq (scRNA-seq) has been widely employed to profile cellular transcriptomes, however, its power of analysing transcription start sites (TSS) has not been fully utilised. Here, we present a computational method suite, CamoTSS, to precisely identify TSS and quantify its expression by leveraging the cDNA on read 1, which enables effective detection of alternative TSS usage. With various experimental data sets, we have demonstrated that CamoTSS can accurately identify TSS and the detected alternative TSS usages showed strong specificity in different biological processes, including cell types across human organs, the development of human thymus, and cancer conditions. As evidenced in nasopharyngeal cancer, alternative TSS usage can also reveal regulatory patterns including systematic TSS dysregulations.
... Indeed, CD34 + -enriched MDS-RS 247 cells displayed a Lin -CD34 -GPA + population averaging ~90% RS/siderocytes which was absent 248in MDS samples without RS(Fig. 4E-G), highlighting important limitations in interpreting 249 magnetically-enriched cell data from MDS-RS.(7)(8)(9)(10) To circumvent these, we evaluated the 250 molecular signature of CD34 transcript-positive HSPC in scRNAseq(Fig. ...
Myelodysplastic syndromes with ring sideroblasts (MDS-RS) commonly develop from hematopoietic stem cells (HSC) bearing mutations in the splicing factor SF3B1 (SF3B1mt). Direct studies into MDS-RS pathobiology have been limited by a lack of model systems that fully recapitulate erythroid biology and RS development and the inability to isolate viable human RS. Here, we combined successful direct RS isolation from patient samples, high-throughput multiomics analysis of cells encompassing the SF3B1mt stem-erythroid continuum, and functional assays to investigate the impact of SF3B1mt on erythropoiesis and RS accumulation. The isolated RS differentiated, egressed into the blood, escaped traditional nonsense-mediated decay (NMD) mechanisms, and leveraged stress-survival pathways that hinder wild-type hematopoiesis through pathogenic GDF15 overexpression. Importantly, RS constituted a contaminant of magnetically enriched CD34+ cells, skewing bulk transcriptomic data. Mis-splicing in SF3B1mt cells was intensified by erythroid differentiation through accelerated RNA splicing and decreased NMD activity, and SF3B1mt led to truncations in several MDS-implicated genes. Finally, RNA mis-splicing induced an uncoupling of RNA and protein expression, leading to critical abnormalities in proapoptotic p53 pathway genes. Overall, this characterization of erythropoiesis in SF3B1mt RS provides a resource for studying MDS-RS and uncovers insights into the unexpectedly active biology of the “dead-end” RS.
Significance
Ring sideroblast isolation combined with state-of-the-art multiomics identifies survival mechanisms underlying SF3B1-mutant erythropoiesis and establishes an active role for erythroid differentiation and ring sideroblasts themselves in SF3B1-mutant myelodysplastic syndrome pathogenesis.
... In addition, a specific subtype of low-risk MDS, namely MDS with ringed sideroblasts, occurs in 20% of all MDS patients. Splicing factor 3 B subunit 1 (SF3B1) mutations in hematopoietic stem and progenitor cells is a hallmark of this disease, inducing aberrant splicing of genes involved in heme biosynthesis and mitochondrial iron transport, leading to the abnormal deposition of iron in erythroblasts, and resulting in dysfunctional hemoglobin synthesis and formation of ringed sideroblasts (Visconte et al., 2015;Dolatshad et al., 2015;Dolatshad et al., 2016;Shiozawa et al., 2018;Clough et al., 2022). Although preclinical data in mouse models predicted a therapeutic effect of splicing inhibition, a recent phase I clinical trial did not yield significant clinical improvement (Lee et al., 2016;Steensma et al., 2021). ...
Erythroblasts possess unique characteristics as they undergo differentiation from hematopoietic stem cells. During terminal erythropoiesis, these cells incorporate large amounts of iron in order to generate hemoglobin and ultimately undergo enucleation to become mature red blood cells, ultimately delivering oxygen in the circulation. Thus, erythropoiesis is a finely tuned, multifaceted process requiring numerous properly timed physiological events to maintain efficient production of 2 million red blood cells per second in steady state. Iron is required for normal functioning in all human cells, the erythropoietic compartment consuming the majority in light of the high iron requirements for hemoglobin synthesis. Recent evidence regarding the crosstalk between erythropoiesis and iron metabolism sheds light on the regulation of iron availability by erythroblasts and the consequences of insufficient as well as excess iron on erythroid lineage proliferation and differentiation. In addition, significant progress has been made in our understanding of dysregulated iron metabolism in various congenital and acquired malignant and non-malignant diseases. Finally, we report several actual as well as theoretical opportunities for translating the recently acquired robust mechanistic understanding of iron metabolism regulation to improve management of patients with disordered erythropoiesis, such as anemia of chronic inflammation, β-thalassemia, polycythemia vera, and myelodysplastic syndromes.
... SF3B1 mutations typically result in aberrant splicing due to the recognition and utilization of cryptic 3 SS. Many of these critically spliced transcripts undergo nonsense-mediated decay affecting gene expression [101,102]. One specific substitution in SF3B1, K700E, found in patients with MDS-RS, has been demonstrated to disrupt its interaction with the SURP and G-patch domain containing 1 (SUGP1) protein. ...
Over the last decade, our understanding of spliceosome structure and function has significantly improved, refining the study of the impact of dysregulated splicing on human disease. As a result, targeted splicing therapeutics have been developed, treating various diseases including spinal muscular atrophy and Duchenne muscular dystrophy. These advancements are very promising and emphasize the critical role of proper splicing in maintaining human health. Herein, we provide an overview of the current information on the composition and assembly of early splicing complexes-commitment complex and pre-spliceosome-and their association with human disease.
