RESUMEN
The auxiliary factor of U2 small nuclear RNA (U2AF) is a heterodimer consisting of 65- and 35-kD proteins that bind the polypyrimidine tract (PPT) and AG dinucleotides at the 3' splice site (3'ss). The gene encoding U2AF35 (U2AF1) is alternatively spliced, giving rise to two isoforms U2AF35a and U2AF35b. Here, we knocked down U2AF35 and each isoform and characterized transcriptomes of HEK293 cells with varying U2AF35/U2AF65 and U2AF35a/b ratios. Depletion of both isoforms preferentially modified alternative RNA processing events without widespread failure to recognize 3'ss or constitutive exons. Over a third of differentially used exons were terminal, resulting largely from the use of known alternative polyadenylation (APA) sites. Intronic APA sites activated in depleted cultures were mostly proximal whereas tandem 3'UTR APA was biased toward distal sites. Exons upregulated in depleted cells were preceded by longer AG exclusion zones and PPTs than downregulated or control exons and were largely activated by PUF60 and repressed by CAPERα. The U2AF(35) repression and activation was associated with a significant interchange in the average probabilities to form single-stranded RNA in the optimal PPT and branch site locations and sequences further upstream. Although most differentially used exons were responsive to both U2AF subunits and their inclusion correlated with U2AF levels, a small number of transcripts exhibited distinct responses to U2AF35a and U2AF35b, supporting the existence of isoform-specific interactions. These results provide new insights into function of U2AF and U2AF35 in alternative RNA processing.
Asunto(s)
Exones , Proteínas Nucleares/genética , Empalme del ARN , Ribonucleoproteínas/genética , Análisis de Secuencia de ARN , Células HEK293 , Humanos , Factor de Empalme U2AFRESUMEN
BACKGROUND: An understanding of linkage disequilibrium (LD) structures in the human genome underpins much of medical genetics and provides a basis for disease gene mapping and investigating biological mechanisms such as recombination and selection. Whole genome sequencing (WGS) provides the opportunity to determine LD structures at maximal resolution. RESULTS: We compare LD maps constructed from WGS data with LD maps produced from the array-based HapMap dataset, for representative European and African populations. WGS provides up to 5.7-fold greater SNP density than array-based data and achieves much greater resolution of LD structure, allowing for identification of up to 2.8-fold more regions of intense recombination. The absence of ascertainment bias in variant genotyping improves the population representativeness of the WGS maps, and highlights the extent of uncaptured variation using array genotyping methodologies. The complete capture of LD patterns using WGS allows for higher genome-wide association study (GWAS) power compared to array-based GWAS, with WGS also allowing for the analysis of rare variation. The impact of marker ascertainment issues in arrays has been greatest for Sub-Saharan African populations where larger sample sizes and substantially higher marker densities are required to fully resolve the LD structure. CONCLUSIONS: WGS provides the best possible resource for LD mapping due to the maximal marker density and lack of ascertainment bias. WGS LD maps provide a rich resource for medical and population genetics studies. The increasing availability of WGS data for large populations will allow for improved research utilising LD, such as GWAS and recombination biology studies.
Asunto(s)
Genética de Población , Genoma Humano , Desequilibrio de Ligamiento/genética , Secuencia de Bases , Frecuencia de los Genes/genética , Marcadores Genéticos , Técnicas de Genotipaje , Humanos , Mapeo Físico de Cromosoma , Tamaño de la MuestraRESUMEN
Rearrangements of chromosome band 9p24 are known to be associated with JAK2 fusion genes, e.g., t(8;9)(p22;p24) with a PCM1-JAK2 and t(9;22)(p24;q11) with a BCR-JAK2 fusion gene, respectively. In association with myeloid neoplasms, the clinical course is aggressive, and in absence of effective conventional treatment options, long-term remission is usually only observed after allogeneic stem cell transplantation (ASCT). With the discovery of inhibitors of the JAK2 tyrosine kinase and based on encouraging in vitro and in vivo data, we treated two male patients with myeloid neoplasms and a PCM1-JAK2 or a BCR-JAK2 fusion gene, respectively, with the JAK1/JAK2 inhibitor ruxolitinib. After 12 months of treatment, both patients achieved a complete clinical, hematologic, and cytogenetic response. Non-hematologic toxicity was only grade 1 while no hematologic toxicity was observed. However, remission in both patients was only short-term, with relapse occurring after 18 and 24 months, respectively, making ASCT indispensable in both cases. This data highlight (1) the ongoing importance of cytogenetic analysis for the diagnostic work-up of myeloid neoplasms as it may guide targeted therapy and (2) remission under ruxolitinib may only be short-termed in JAK2 fusion genes but it may be an important bridging therapy prior to ASCT.
Asunto(s)
Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Proteínas de Fusión Oncogénica/genética , Pirazoles/uso terapéutico , Secuencia de Aminoácidos , Secuencia de Bases , Análisis Mutacional de ADN , Humanos , Hibridación Fluorescente in Situ , Quinasas Janus/antagonistas & inhibidores , Masculino , Datos de Secuencia Molecular , Recurrencia Local de Neoplasia , Nitrilos , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas c-bcr/genética , Pirimidinas , Inducción de Remisión , Factores de TiempoAsunto(s)
Autoantígenos/genética , Proteínas de Ciclo Celular/genética , Síndrome Hipereosinofílico/genética , Leucemia Mielomonocítica Crónica/genética , Proteínas de Fusión Oncogénica/genética , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/genética , Antineoplásicos/uso terapéutico , Autoantígenos/química , Proteínas de Ciclo Celular/química , Cromosomas Humanos Par 5/genética , Cromosomas Humanos Par 8/genética , Humanos , Síndrome Hipereosinofílico/tratamiento farmacológico , Mesilato de Imatinib/uso terapéutico , Leucemia Mielomonocítica Crónica/tratamiento farmacológico , Masculino , Persona de Mediana Edad , Mutagénesis Insercional , Proteínas de Fusión Oncogénica/química , Inhibidores de Proteínas Quinasas/uso terapéutico , ARN Neoplásico/genética , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/químicaRESUMEN
ATM is an important cancer susceptibility gene that encodes a critical apical kinase of the DNA damage response (DDR) pathway. We show that a key nonsense-mediated RNA decay switch exon (NSE) in ATM is repressed by U2AF, PUF60 and hnRNPA1. The NSE activation was haplotype-specific and was most promoted by cytosine at rs609621 in the NSE 3' splice-site (3'ss), which is predominant in high cancer risk populations. NSE levels were deregulated in leukemias and were influenced by the identity of U2AF35 residue 34. We also identify splice-switching oligonucleotides (SSOs) that exploit competition of adjacent pseudoexons to modulate NSE levels. The U2AF-regulated exon usage in the ATM signalling pathway was centred on the MRN/ATM-CHEK2-CDC25-cdc2/cyclin-B axis and preferentially involved transcripts implicated in cancer-associated gene fusions and chromosomal translocations. These results reveal important links between 3'ss control and ATM-dependent responses to double-strand DNA breaks, demonstrate functional plasticity of intronic variants and illustrate versatility of intronic SSOs that target pseudo-3'ss to modify gene expression.