RESUMEN
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.
Asunto(s)
Bases de Datos Genéticas , Evolución Molecular , Intrones/genética , Empalme del ARN/genética , Empalmosomas/genética , Animales , Genoma , Humanos , Filogenia , Plantas/genética , ARN Largo no Codificante/genética , ARN Nuclear Pequeño/genética , Ribonucleoproteínas Nucleares Pequeñas/genética , Levaduras/genéticaRESUMEN
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.
Asunto(s)
Proliferación Celular/fisiología , Factor de Transcripción E2F1/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo C/fisiología , Transcripción Genética/fisiología , Ciclo Celular/genética , Factor de Transcripción E2F1/genética , Silenciador del Gen , Células HeLa , Ribonucleoproteína Heterogénea-Nuclear Grupo C/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo C/metabolismo , Humanos , Unión Proteica , ARN Polimerasa II/metabolismo , ARN Mensajero/metabolismo , Transcripción Genética/genética , TranscriptomaRESUMEN
Core RNA-processing reactions in eukaryotic cells occur cotranscriptionally in a chromatin context, but the relationship between chromatin structure and pre-mRNA processing is poorly understood. We observed strong nucleosome depletion around human polyadenylation sites (PAS) and nucleosome enrichment just downstream of PAS. In genes with multiple alternative PAS, higher downstream nucleosome affinity was associated with higher PAS usage, independently of known PAS motifs that function at the RNA level. Conversely, exons were associated with distinct peaks in nucleosome density. Exons flanked by long introns or weak splice sites exhibited stronger nucleosome enrichment, and incorporation of nucleosome density data improved splicing simulation accuracy. Certain histone modifications, including H3K36me3 and H3K27me2, were specifically enriched on exons, suggesting active marking of exon locations at the chromatin level. Together, these findings provide evidence for extensive functional connections between chromatin structure and RNA processing.
Asunto(s)
Cromatina/genética , Exones/genética , Poliadenilación/genética , Composición de Base/genética , Histonas/metabolismo , Humanos , Intrones/genética , Metilación , Nucleosomas/metabolismo , Sitios de Empalme de ARN/genética , Empalme del ARN/genética , Secuencias Reguladoras de Ácidos Nucleicos/genéticaRESUMEN
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.
Asunto(s)
Enanismo/genética , Retardo del Crecimiento Fetal/genética , Microcefalia/genética , Osteocondrodisplasias/genética , ARN Nuclear Pequeño/genética , Empalmosomas/genética , Animales , Secuencia de Bases , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Enanismo/metabolismo , Enanismo/patología , Retardo del Crecimiento Fetal/metabolismo , Retardo del Crecimiento Fetal/patología , Perfilación de la Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Recién Nacido , Microcefalia/metabolismo , Microcefalia/patología , Datos de Secuencia Molecular , Mutación , Conformación de Ácido Nucleico , Osteocondrodisplasias/metabolismo , Osteocondrodisplasias/patología , Unión Proteica , ARN Nuclear Pequeño/química , ARN Nuclear Pequeño/metabolismo , Empalmosomas/química , Empalmosomas/fisiologíaRESUMEN
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.
Asunto(s)
Empalme del ARN , Animales , Humanos , Intrones , Mutación , Neoplasias/genética , Empalmosomas/genética , Empalmosomas/metabolismoRESUMEN
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.
Asunto(s)
Transformación Celular Neoplásica/genética , Mutación , Proteínas Nucleares/genética , Fosfoproteínas/genética , Empalme del ARN/genética , Ribonucleoproteína Nuclear Pequeña U2/genética , Ribonucleoproteínas/genética , Secuencia de Bases , Femenino , Estudios de Asociación Genética , Humanos , Leucemia Mieloide/diagnóstico , Leucemia Mieloide/genética , Leucemia Mieloide/mortalidad , Masculino , Tasa de Mutación , Síndromes Mielodisplásicos/diagnóstico , Síndromes Mielodisplásicos/genética , Síndromes Mielodisplásicos/mortalidad , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Pronóstico , Factores de Empalme de ARN , Ribonucleoproteína Nuclear Pequeña U2/metabolismo , Ribonucleoproteínas/metabolismo , Alineación de Secuencia , Factores de Empalme Serina-Arginina , Empalmosomas/genética , Empalmosomas/metabolismo , Factor de Empalme U2AFRESUMEN
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.
Asunto(s)
Anemia Sideroblástica/patología , Biomarcadores de Tumor/genética , Haploinsuficiencia , Mutación/genética , Síndromes Mielodisplásicos/patología , Fosfoproteínas/metabolismo , Fosfoproteínas/fisiología , Ribonucleoproteína Nuclear Pequeña U2/metabolismo , Ribonucleoproteína Nuclear Pequeña U2/fisiología , Adolescente , Adulto , Anciano , Anemia Sideroblástica/etiología , Anemia Sideroblástica/metabolismo , Animales , Biomarcadores de Tumor/metabolismo , Células Cultivadas , Femenino , Perfilación de la Expresión Génica , Humanos , Células K562 , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Síndromes Mielodisplásicos/etiología , Síndromes Mielodisplásicos/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Fosfoproteínas/genética , Factores de Empalme de ARN , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ribonucleoproteína Nuclear Pequeña U2/genética , Adulto JovenRESUMEN
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.
RESUMEN
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.
Asunto(s)
Transformación Celular Neoplásica/genética , Neoplasias Hematológicas/genética , Empalmosomas/genética , Humanos , Leucemia/genética , Mutación , Empalme del ARN/genética , ARN Neoplásico/genética , Transducción de Señal/genéticaAsunto(s)
Intrones , Empalmosomas , Humanos , Proteínas Matrilinas/genética , Filogenia , ARN Nuclear Pequeño/genéticaRESUMEN
U6 and U6atac snRNAs play analogous critical roles in the major U2-dependent and minor U12-dependent spliceosomes, respectively. Previous results have shown that most of the functional cores of these two snRNAs are either highly similar in sequence or functionally interchangeable. Thus, a mechanism must exist to restrict each snRNA to its own spliceosome. Here we show that a chimeric U6 snRNA containing the unique and highly conserved 3' end domain of U6atac snRNA is able to function in vivo in U12-dependent spliceosomal splicing. Function of this chimera required the coexpression of a modified U4atac snRNA; U4 snRNA could not substitute. Partial deletions of this element in vivo, as well as in vitro antisense experiments, showed that the 3' end domain of U6atac snRNA is necessary to direct the U4atac/U6atac.U5 tri-snRNP to the forming U12-dependent spliceosome. In vitro experiments also uncovered a role for U4atac snRNA in this targeting.
Asunto(s)
ARN Nuclear Pequeño/química , Empalmosomas/metabolismo , Secuencia de Bases , Secuencia Conservada , Humanos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Empalme del ARN , ARN Nuclear Pequeño/metabolismo , TransfecciónRESUMEN
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.
Asunto(s)
Leucemia Mieloide/genética , Síndromes Mielodisplásicos/genética , Proteínas Nucleares/genética , Empalme del ARN , Proteínas de Unión al ARN/genética , Secuencia de Bases , Exones , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Intrones , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patología , Mutación , Síndromes Mielodisplásicos/metabolismo , Síndromes Mielodisplásicos/patología , Proteínas Nucleares/metabolismo , ARN Nuclear Pequeño/genética , ARN Nuclear Pequeño/metabolismo , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteína Nuclear Pequeña U1/genética , Ribonucleoproteína Nuclear Pequeña U1/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de Señal , EmpalmosomasRESUMEN
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.
Asunto(s)
ARN Helicasas DEAD-box , Síndromes Mielodisplásicos , Animales , ARN Helicasas DEAD-box/genética , Células Germinativas , Hematopoyesis/genética , Ratones , Mutación/genética , Síndromes Mielodisplásicos/genéticaRESUMEN
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.
Asunto(s)
Embrión no Mamífero/citología , Células Madre Hematopoyéticas/citología , Estructuras R-Loop , Proteínas de Pez Cebra/metabolismo , Animales , Animales Modificados Genéticamente , Diferenciación Celular , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Embrión no Mamífero/metabolismo , Células Madre Hematopoyéticas/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Transducción de Señal , Pez Cebra , Proteínas de Pez Cebra/genéticaRESUMEN
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.
Asunto(s)
Empalme Alternativo , Biomarcadores de Tumor , Regulación Neoplásica de la Expresión Génica , Trastornos Mieloproliferativos/genética , Médula Ósea/patología , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Estudios de Casos y Controles , Deleción Cromosómica , Análisis por Conglomerados , Susceptibilidad a Enfermedades , Perfilación de la Expresión Génica , Humanos , Mutación con Pérdida de Función , Mutación , Síndromes Mielodisplásicos/diagnóstico , Síndromes Mielodisplásicos/genética , Síndromes Mielodisplásicos/terapia , Trastornos Mieloproliferativos/diagnóstico , Trastornos Mieloproliferativos/terapia , Factores de Empalme de ARN/genética , Factores de Empalme de ARN/metabolismo , TranscriptomaRESUMEN
The X-ray crystal structure of an excised group II self-splicing intron was recently solved by the Pyle group. Here we review some of the notable features of this structure and what they may tell us about the catalytic active site of the group II ribozyme and potentially the spliceosome. The new structure validates the central role of domain V in both the structure and catalytic function of the ribozyme and resolves several outstanding puzzles raised by previous biochemical, genetic and structural studies. While lacking both exons as well as the cleavage sites and nucleophiles, the structure reveals how a network of tertiary interactions can position two divalent metal ions in a configuration that is ideal for catalysis.
Asunto(s)
Empalme Alternativo , Intrones , ARN Catalítico/química , Empalmosomas/química , Empalmosomas/enzimología , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Iones/química , Metales/química , Conformación de Ácido Nucleico , ARN Catalítico/clasificaciónRESUMEN
Highly conserved sequences at the 5' splice site and branch site of U12-dependent introns are important determinants for splicing by U12-dependent spliceosomes. This study investigates the in vivo splicing phenotypes of mutations in the branch site consensus sequence of the U12-dependent intron F from a human NOL1 (P120) minigene. Intron F contains a fully consensus branch site sequence (UUCCUUAAC). Mutations at each position were analyzed for their effects on U12-dependent splicing in vivo. Mutations at most positions resulted in a significant reduction of correct U12-dependent splicing. Defects observed included increased unspliced RNA levels, the activation of cryptic U2-dependent 5' and 3' splice sites, and the activation of cryptic U12-dependent branch/3' splice sites. A strong correlation was observed between the predicted thermodynamic stability of the branch site: U12 snRNA interaction and correct U12-dependent splicing. The lack of a polypyrimidine tract between the branch site and 3' splice site of U12-dependent introns and the observed reliance on base-pairing interactions for correct U12-dependent splicing emphasize the importance of RNA/RNA interactions during U12-dependent intron recognition and proper splice site selection.
Asunto(s)
Empalme Alternativo , Intrones , Sitios de Empalme de ARN , ARN Nuclear Pequeño/metabolismo , Secuencia de Bases , Secuencia de Consenso , Humanos , Mutación , Proteínas Nucleares/genética , Sitios de Empalme de ARN/genética , ARN Nuclear Pequeño/genética , Empalmosomas/genética , Empalmosomas/metabolismo , ARNt MetiltransferasasRESUMEN
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.
Asunto(s)
Mutación , Factores de Empalme de ARN , Empalme del ARN/genética , Empalmosomas , Humanos , Síndromes Mielodisplásicos/genética , Síndromes Mielodisplásicos/metabolismo , Factores de Empalme de ARN/genética , Factores de Empalme de ARN/metabolismo , Empalmosomas/genética , Empalmosomas/metabolismoRESUMEN
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.