A multimodal analysis of genomic and RNA splicing features in myeloid malignancies.

RNA splicing dysfunctions are more widespread than what is believed by only estimating the effects resulting by splicing factor mutations (SFMT) in myeloid neoplasia (MN). The genetic complexity of MN is amenable to machine learning (ML) strategies. We applied an integrative ML approach to identify co-varying features by combining genomic lesions (mutations, deletions, and copy number), exon-inclusion ratio as measure of RNA splicing (percent spliced in, PSI), and gene expression (GE) of 1,258 MN and 63 normal controls. We identified 15 clusters based on mutations, GE, and PSI. Different PSI levels were present at various extents regardless of SFMT suggesting that changes in RNA splicing were not strictly related to SFMT. Combination of PSI and GE further distinguished the features and identified PSI similarities and differences, common pathways, and expression signatures across clusters. Thus, multimodal features can resolve the complex architecture of MN and help identifying convergent molecular and transcriptomic pathways amenable to therapies.

Germline DDX41 mutations cause ineffective hematopoiesis and myelodysplasia.

DDX41 mutations are the most common germline alterations in adult myelodysplastic syndromes (MDSs). The majority of affected individuals harbor germline monoallelic frameshift DDX41 mutations and subsequently acquire somatic mutations in their other DDX41 allele, typically missense R525H. Hematopoietic progenitor cells (HPCs) with biallelic frameshift and R525H mutations undergo cell cycle arrest and apoptosis, causing bone marrow failure in mice. Mechanistically, DDX41 is essential for small nucleolar RNA (snoRNA) processing, ribosome assembly, and protein synthesis. Although monoallelic DDX41 mutations do not affect hematopoiesis in young mice, a subset of aged mice develops features of MDS. Biallelic mutations in DDX41 are observed at a low frequency in non-dominant hematopoietic stem cell clones in bone marrow (BM) from individuals with MDS. Mice chimeric for monoallelic DDX41 mutant BM cells and a minor population of biallelic mutant BM cells develop hematopoietic defects at a younger age, suggesting that biallelic DDX41 mutant cells are disease modifying in the context of monoallelic DDX41 mutant BM.

Functional analyses of human LUC7-like proteins involved in splicing regulation and myeloid neoplasms.

Vertebrates have evolved three paralogs, termed LUC7L, LUC7L2, and LUC7L3, of the essential yeast U1 small nuclear RNA (snRNA)-associated splicing factor Luc7p. We investigated the mechanistic and regulatory functions of these putative splicing factors, of which one (LUC7L2) is mutated or deleted in myeloid neoplasms. Protein interaction data show that all three proteins bind similar core but distinct regulatory splicing factors, probably mediated through their divergent arginine-serine-rich domains, which are not present in Luc7p. Knockdown of each factor reveals mostly unique sets of significantly dysregulated alternative splicing events dependent on their binding locations, which are largely non-overlapping. Notably, knockdown of LUC7L2 alone significantly upregulates the expression of multiple spliceosomal factors and downregulates glycolysis genes, possibly contributing to disease pathogenesis. RNA binding studies reveal that LUC7L2 and LUC7L3 crosslink to weak 5' splice sites and to the 5' end of U1 snRNA, establishing an evolutionarily conserved role in 5' splice site selection.

Excessive R-loops trigger an inflammatory cascade leading to increased HSPC production.

Hematopoietic stem and progenitor cells (HSPCs) arise during embryonic development and are essential for sustaining the blood and immune systems throughout life. Tight regulation of HSPC numbers is critical for hematopoietic homeostasis. Here, we identified DEAD-box helicase 41 (Ddx41) as a gatekeeper of HSPC production. Using zebrafish ddx41 mutants, we unveiled a critical role for this helicase in regulating HSPC production at the endothelial-to-hematopoietic transition. We determined that Ddx41 suppresses the accumulation of R-loops, nucleic acid structures consisting of RNA:DNA hybrids and ssDNAs whose equilibrium is essential for cellular fitness. Excess R-loop levels in ddx41 mutants triggered the cGAS-STING inflammatory pathway leading to increased numbers of hemogenic endothelium and HSPCs. Elevated R-loop accumulation and inflammatory signaling were observed in human cells with decreased DDX41, suggesting possible conservation of mechanism. These findings delineate that precise regulation of R-loop levels during development is critical for limiting cGAS-STING activity and HSPC numbers.

Complex landscape of alternative splicing in myeloid neoplasms.

Myeloid neoplasms are characterized by frequent mutations in at least seven components of the spliceosome that have distinct roles in the process of pre-mRNA splicing. Hotspot mutations in SF3B1, SRSF2, U2AF1 and loss of function mutations in ZRSR2 have revealed widely different aberrant splicing signatures with little overlap. However, previous studies lacked the power necessary to identify commonly mis-spliced transcripts in heterogeneous patient cohorts. By performing RNA-Seq on bone marrow samples from 1258 myeloid neoplasm patients and 63 healthy bone marrow donors, we identified transcripts frequently mis-spliced by mutated splicing factors (SF), rare SF mutations with common alternative splicing (AS) signatures, and SF-dependent neojunctions. We characterized 17,300 dysregulated AS events using a pipeline designed to predict the impact of mis-splicing on protein function. Meta-splicing analysis revealed a pattern of reduced levels of retained introns among disease samples that was exacerbated in patients with splicing factor mutations. These introns share characteristics with "detained introns," a class of introns that have been shown to promote differentiation by detaining pro-proliferative transcripts in the nucleus. In this study, we have functionally characterized 17,300 targets of mis-splicing by the SF mutations, identifying a common pathway by which AS may promote maintenance of a proliferative state.

Spliceosomal factor mutations and mis-splicing in MDS.

Somatic, heterozygous missense and nonsense mutations in at least seven proteins that function in the spliceosome are found at high frequency in MDS patients. These proteins act at various steps in the process of splicing by the spliceosome and lead to characteristic alterations in the alternative splicing of a subset of genes. Several studies have investigated the effects of these mutations and have attempted to identify a commonly affected gene or pathway. Here, we summarize what is known about the normal function of these proteins and how the mutations alter the splicing landscape of the genome. We also summarize the commonly mis-spliced gene targets and discuss the state of mechanistic unification that has been achieved. Finally, we discuss alternative mechanisms by which these mutations may lead to disease.

Clinical interpretation of variants identified in RNU4ATAC, a non-coding spliceosomal gene.

Biallelic variants in RNU4ATAC, a non-coding gene transcribed into the minor spliceosome component U4atac snRNA, are responsible for three rare recessive developmental diseases, namely Taybi-Linder/MOPD1, Roifman and Lowry-Wood syndromes. Next-generation sequencing of clinically heterogeneous cohorts (children with either a suspected genetic disorder or a congenital microcephaly) recently identified mutations in this gene, illustrating how profoundly these technologies are modifying genetic testing and assessment. As RNU4ATAC has a single non-coding exon, the bioinformatic prediction algorithms assessing the effect of sequence variants on splicing or protein function are irrelevant, which makes variant interpretation challenging to molecular diagnostic laboratories. In order to facilitate and improve clinical diagnostic assessment and genetic counseling, we present i) an update of the previously reported RNU4ATAC mutations and an analysis of the genetic variations affecting this gene using the Genome Aggregation Database (gnomAD) resource; ii) the pathogenicity prediction performances of scores computed based on an RNA structure prediction tool and of those produced by the Combined Annotation Dependent Depletion tool for the 285 RNU4ATAC variants identified in patients or in large-scale sequencing projects; iii) a method, based on a cellular assay, that allows to measure the effect of RNU4ATAC variants on splicing efficiency of a minor (U12-type) reporter intron. Lastly, the concordance of bioinformatic predictions and cellular assay results was investigated.

Comprehensive database and evolutionary dynamics of U12-type introns.

During nuclear maturation of most eukaryotic pre-messenger RNAs and long non-coding RNAs, introns are removed through the process of RNA splicing. Different classes of introns are excised by the U2-type or the U12-type spliceosomes, large complexes of small nuclear ribonucleoprotein particles and associated proteins. We created intronIC, a program for assigning intron class to all introns in a given genome, and used it on 24 eukaryotic genomes to create the Intron Annotation and Orthology Database (IAOD). We then used the data in the IAOD to revisit several hypotheses concerning the evolution of the two classes of spliceosomal introns, finding support for the class conversion model explaining the low abundance of U12-type introns in modern genomes.

The hnRNP RALY regulates transcription and cell proliferation by modulating the expression of specific factors including the proliferation marker E2F1.

The heterogeneous nuclear ribonucleoproteins (hnRNP) form a large family of RNA-binding proteins that exert numerous functions in RNA metabolism. RALY is a member of the hnRNP family that binds poly-U-rich elements within several RNAs and regulates the expression of specific transcripts. RALY is up-regulated in different types of cancer, and its down-regulation impairs cell cycle progression. However, the RALY's role in regulating RNA levels remains elusive. Here, we show that numerous genes coding for factors involved in transcription and cell cycle regulation exhibit an altered expression in RALY-down-regulated HeLa cells, consequently causing impairments in transcription, cell proliferation, and cell cycle progression. Interestingly, by comparing the list of RALY targets with the list of genes affected by RALY down-regulation, we found an enrichment of RALY mRNA targets in the down-regulated genes upon RALY silencing. The affected genes include the E2F transcription factor family. Given its role as proliferation-promoting transcription factor, we focused on E2F1. We demonstrate that E2F1 mRNA stability and E2F1 protein levels are reduced in cells lacking RALY expression. Finally, we also show that RALY interacts with transcriptionally active chromatin in both an RNA-dependent and -independent manner and that this association is abolished in the absence of active transcription. Taken together, our results highlight the importance of RALY as an indirect regulator of transcription and cell cycle progression through the regulation of specific mRNA targets, thus strengthening the possibility of a direct gene expression regulation exerted by RALY.

DDX41-related myeloid neoplasia.

While early presentation of familial leukemia syndromes is typical, long disease anticipation may mask cases of familial traits in seemingly spontaneous disease. Germline mutations in DDX41 gene have been discovered in several leukemia families, as well as in mostly adult patients with seemingly spontaneous disease but having strong family histories of myeloid neoplasia. As with other familial genes, DDX41 mutation carriers can develop neoplasia through acquisition of another somatic mutation, thereby affecting both DDX41 alleles. In other patients, somatic mutations of different driver genes can substitute for acquired missense DDX41 during progression. Conversely, non-familial cases with heterozygous somatic DDX41 mutations point towards other mutations that can substitute for the germ line founder DDX41 lesions. In either circumstance, total inactivation of DDX41 appears to be cell-lethal, explaining why frameshift germline lesions have not been found to be accompanied by deletions of the DDX41 locus on 5q. The precise function of the DDX41 protein is unknown; considerable evidence suggests its involvement in RNA splicing. Thus DDX41 can be included in the now large group of mutated spliceosomal genes affected in myeloid neoplasia. However, it appears that DDX4 is so far the only example of a germline spliceosomal mutation in leukemia. Clinically, recognition of DDX41 mutated cases may have implications for surveillance, assessment of prognosis, and, perhaps, for design of targeted therapies.

Inherited and Somatic Defects in DDX41 in Myeloid Neoplasms.

Most cases of adult myeloid neoplasms are routinely assumed to be sporadic. Here, we describe an adult familial acute myeloid leukemia (AML) syndrome caused by germline mutations in the DEAD/H-box helicase gene DDX41. DDX41 was also found to be affected by somatic mutations in sporadic cases of myeloid neoplasms as well as in a biallelic fashion in 50% of patients with germline DDX41 mutations. Moreover, corresponding deletions on 5q35.3 present in 6% of cases led to haploinsufficient DDX41 expression. DDX41 lesions caused altered pre-mRNA splicing and RNA processing. DDX41 is exemplary of other RNA helicase genes also affected by somatic mutations, suggesting that they constitute a family of tumor suppressor genes.

Biochemical defects in minor spliceosome function in the developmental disorder MOPD I.

Biallelic mutations of the human RNU4ATAC gene, which codes for the minor spliceosomal U4atac snRNA, cause the developmental disorder, MOPD I/TALS. To date, nine separate mutations in RNU4ATAC have been identified in MOPD I patients. Evidence suggests that all of these mutations lead to abrogation of U4atac snRNA function and impaired minor intron splicing. However, the molecular basis of these effects is unknown. Here, we use a variety of in vitro and in vivo assays to address this question. We find that only one mutation, 124G>A, leads to significantly reduced expression of U4atac snRNA, whereas four mutations, 30G>A, 50G>A, 50G>C and 51G>A, show impaired binding of essential protein components of the U4atac/U6atac di-snRNP in vitro and in vivo. Analysis of MOPD I patient fibroblasts and iPS cells homozygous for the most common mutation, 51G>A, shows reduced levels of the U4atac/U6atac.U5 tri-snRNP complex as determined by glycerol gradient sedimentation and immunoprecipitation. In this report, we establish a mechanistic basis for MOPD I disease and show that the inefficient splicing of genes containing U12-dependent introns in patient cells is due to defects in minor tri-snRNP formation, and the MOPD I-associated RNU4ATAC mutations can affect multiple facets of minor snRNA function.

SF3B1 haploinsufficiency leads to formation of ring sideroblasts in myelodysplastic syndromes.

Whole exome/genome sequencing has been fundamental in the identification of somatic mutations in the spliceosome machinery in myelodysplastic syndromes (MDSs) and other hematologic disorders. SF3B1, splicing factor 3b subunit 1 is mutated in 60%-80% of refractory anemia with ring sideroblasts (RARS) and RARS associated with thrombocytosis (RARS-T), 2 distinct subtypes of MDS and MDS/myeloproliferative neoplasms (MDSs/MPNs). An idiosyncratic feature of RARS/RARS-T is the presence of abnormal sideroblasts characterized by iron overload in the mitochondria, called RS. Based on the high frequency of mutations of SF3B1 in RARS/RARS-T, we investigated the consequences of SF3B1 alterations. Ultrastructurally, SF3B1 mutants showed altered iron distribution characterized by coarse iron deposits compared with wild-type RARS patients by transmission electron microscopy. SF3B1 knockdown experiments in K562 cells resulted in down-regulation of U2-type intron-splicing by RT-PCR. RNA-sequencing analysis of SF3B1 mutants showed differentially used genes relevant in MDS pathogenesis, such as ASXL1, CBL, EZH, and RUNX families. A SF3B pharmacologic inhibitor, meayamycin, induced the formation of RS in healthy BM cells. Further, BM aspirates of Sf3b1 heterozygous knockout mice showed RS by Prussian blue. In conclusion, we report the first experimental evidence of the association between SF3B1 and RS phenotype. Our data suggest that SF3B1 haploinsufficiency leads to RS formation.

Defects in spliceosomal machinery: a new pathway of leukaemogenesis.

Proper splicing of pre-mRNA is required for protein synthesis and therefore is a fundamental cellular function. The discovery of a variety of somatic spliceosomal mutations in haematological malignancies, including myeloid neoplasms and chronic lymphocytic leukaemia has pointed to a new leukaemogenic pathway involving spliceosomal dysfunction. Theoretically, spliceosomal mutations can lead to activation of incorrect splice sites, intron retention or aberrant alternative splicing occurring in patterns generated by mutations of individual spliceosomal proteins. Such events can produce a defective balance between protein isoforms leading to functional consequences including defective regulation of proliferation and differentiation. The observed pattern of occurrence of highly specific missense mutations, coupled with the lack of nonsense mutations and deletions, implies a gain-of-function or better gain-of-dysfunction mechanism. Incorrect splicing of downstream genes, such as tumour suppressor genes, may result in haploinsufficient expression through nonsense-mediated mRNA decay. Thus, spliceosomal mutations may, depending on the pattern of affected proteins, lead to similar functional effects on tumour suppressor genes as chromosomal deletions, epigenetic silencing or inactivating/hypomorphic mutations. The prognostic value of the most common mutations and their phenotypic association in the clinical setting is currently under investigation. It is likely that spliceosomal mutations may indicate sensitivity to spliceosome inhibitors applied in the form of a synthetic lethal approach. This review discusses the most current aspects of spliceosomal research in the context of haematological malignancies.

New connections between splicing and human disease.

The removal by splicing of introns from the primary transcripts of most mammalian genes is an essential step in gene expression. Splicing is performed by large, complex ribonucleoprotein particles termed spliceosomes. Mammals contain two types that splice out mutually exclusive types of introns. However, the role of the minor spliceosome has been poorly studied. Recent reports have now shown that mutations in one minor spliceosomal snRNA, U4atac, are linked to a rare autosomal recessive developmental defect. In addition, very exciting recent results of exome deep-sequencing have found that recurrent, somatic, heterozygous mutations of other splicing factors occur at high frequencies in particular cancers and pre-cancerous conditions, suggesting that alterations in the core splicing machinery can contribute to tumorigenesis. Mis-splicing of crucial genes may underlie the pathologies of all of these diseases. Identifying these genes and understanding the mechanisms involved in their mis-splicing may lead to advancements in diagnosis and treatment.

Mutations in the spliceosome machinery, a novel and ubiquitous pathway in leukemogenesis.

Myelodysplastic syndromes (MDSs) are chronic and often progressive myeloid neoplasms associated with remarkable heterogeneity in the histomorphology and clinical course. Various somatic mutations are involved in the pathogenesis of MDS. Recently, mutations in a gene encoding a spliceosomal protein, SF3B1, were discovered in a distinct form of MDS with ring sideroblasts. Whole exome sequencing of 15 patients with myeloid neoplasms was performed, and somatic mutations in spliceosomal genes were identified. Sanger sequencing of 310 patients was performed to assess phenotype/genotype associations. To determine the functional effect of spliceosomal mutations, we evaluated pre-mRNA splicing profiles by RNA deep sequencing. We identified additional somatic mutations in spliceosomal genes, including SF3B1, U2AF1, and SRSF2. These mutations alter pre-mRNA splicing patterns. SF3B1 mutations are prevalent in low-risk MDS with ring sideroblasts, whereas U2AF1 and SRSF2 mutations are frequent in chronic myelomonocytic leukemia and advanced forms of MDS. SF3B1 mutations are associated with a favorable prognosis, whereas U2AF1 and SRSF2 mutations are predictive for shorter survival. Mutations affecting spliceosomal genes that result in defective splicing are a new leukemogenic pathway. Spliceosomal genes are probably tumor suppressors, and their mutations may constitute diagnostic biomarkers that could potentially serve as therapeutic targets.

Mutations in U4atac snRNA, a component of the minor spliceosome, in the developmental disorder MOPD I.

Small nuclear RNAs (snRNAs) are essential factors in messenger RNA splicing. By means of homozygosity mapping and deep sequencing, we show that a gene encoding U4atac snRNA, a component of the minor U12-dependent spliceosome, is mutated in individuals with microcephalic osteodysplastic primordial dwarfism type I (MOPD I), a severe developmental disorder characterized by extreme intrauterine growth retardation and multiple organ abnormalities. Functional assays showed that mutations (30G>A, 51G>A, 55G>A, and 111G>A) associated with MOPD I cause defective U12-dependent splicing. Endogenous U12-dependent but not U2-dependent introns were found to be poorly spliced in MOPD I patient fibroblast cells. The introduction of wild-type U4atac snRNA into MOPD I cells enhanced U12-dependent splicing. These results illustrate the critical role of minor intron splicing in human development.