A novel tissue specific alternative splicing variant mitigates phenotypes in Ets2 frame-shift mutant models.
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ABSTRACT:
SUBMITTER: Kishimoto Y
PROVIDER: S-EPMC8050053 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Mutations in splicing factors are recurrent somatic alterations identified in myelodysplastic syndromes (MDS) and they frequently coincide with mutations in epigenetic factors. About 25% of patients present concurrent mutations in such pathways, suggesting a cooperative role in the pathogenesis of MDS. We focused on the splicing factor U2AF1 involved in the recognition of the 3' splice site during pre-mRNA splicing. Using a CRISPR/Cas9 system, we created heterozygous mice with a carboxy-terminal truncated U2af1 allele (U2af1mut/+), studied the U2af1mut/+ hematopoietic system, and did not observe any gross differences in both young (12-13 weeks) and old (23 months) U2af1mut/+ mice, except for a reduction in size of approximately 20%. However, hematopoietic stem/progenitor cells lacked reconstitution capacity in transplantation assays and displayed an aberrant RNA splicing by RNA sequencing. We also evaluated U2af1mut/+ in conjunction with Tet2-deficiency. Novel double mutant U2af1mut/+Tet2-/- mice showed increased monogranulocytic precursors. Hematopoietic stem/progenitor cells were also enhanced and presented functional and transcriptomic alterations. Nonetheless, U2af1mut/+Tet2-/- mice did not succumb to MDS disease over a 6-month observation period. Collectively, our data suggest that cooperation between mutant U2af1 and Tet2 loss is not sufficient for MDS initiation in mice.
| S-EPMC8269301 | biostudies-literature
Project description:MotivationAdvancements in sequencing technologies have highlighted the role of alternative splicing (AS) in increasing transcriptome complexity. This role of AS, combined with the relation of aberrant splicing to malignant states, motivated two streams of research, experimental and computational. The first involves a myriad of techniques such as RNA-Seq and CLIP-Seq to identify splicing regulators and their putative targets. The second involves probabilistic models, also known as splicing codes, which infer regulatory mechanisms and predict splicing outcome directly from genomic sequence. To date, these models have utilized only expression data. In this work, we address two related challenges: Can we improve on previous models for AS outcome prediction and can we integrate additional sources of data to improve predictions for AS regulatory factors.ResultsWe perform a detailed comparison of two previous modeling approaches, Bayesian and Deep Neural networks, dissecting the confounding effects of datasets and target functions. We then develop a new target function for AS prediction in exon skipping events and show it significantly improves model accuracy. Next, we develop a modeling framework that leverages transfer learning to incorporate CLIP-Seq, knockdown and over expression experiments, which are inherently noisy and suffer from missing values. Using several datasets involving key splice factors in mouse brain, muscle and heart we demonstrate both the prediction improvements and biological insights offered by our new models. Overall, the framework we propose offers a scalable integrative solution to improve splicing code modeling as vast amounts of relevant genomic data become available.Availability and implementationCode and data available at: majiq.biociphers.org/jha_et_al_2017/.Contactyosephb@upenn.edu.Supplementary informationSupplementary data are available at Bioinformatics online.
| S-EPMC5870723 | biostudies-literature
Project description:BackgroundArrhythmogenic cardiomyopathy (AC) is one of the leading causes for sudden cardiac death (SCD). Recent studies have identified mutations in cardiac desmosomes as key players in the pathogenesis of AC. However, the specific etiology in individual families remains largely unknown.MethodsA 4-generation family presenting with syncope, lethal ventricular arrhythmia and SCD was recruited. Targeted next generation sequencing (NGS) was performed and validated by Sanger sequencing. Plasmids containing the mutation and wild type (WT) were constructed. Real-time PCR, western-blot and immunofluorescence were performed to detect the functional change due to the mutation.ResultsThe proband, a 56-year-old female, presented with recurrent palpitations and syncope. An ICD was implanted due to her family history of SCD/ aborted SCD. NGS revealed a novel heterozygous frame-shift variant (c.832delG) in Desmoplakin (DSP) among 5 family members. The variant led to frame-shift and premature termination, producing a truncated protein. Cardiac magnetic resonance (CMR) of the family members carrying the same variant shown myocardium thinning and fatty infiltration in the right ventricular, positive bi-ventricular late gadolinium enhancement and severe RV dysfunction, fulfilling the diagnostic criteria of AC. HEK293T cells transfected with mutant plasmids expressed truncated DSP mRNA and protein, upregulation of nuclear junction plakoglobin (JUP) and downregulation of ?-catenin, when compared with WT.ConclusionWe infer that the novel c.832delG variant in DSP was associated with AC in this family, likely through Wnt/?-catenin signaling pathway.
| S-EPMC7011609 | biostudies-literature
Project description:Clusterin (CLU) is one of the risk genes most associated with late onset Alzheimer's disease (AD), and several genetic variants in CLU are associated with AD risk. However, the functional role of known AD risk genetic variants in CLU has been little explored. We investigated the effect of an AD risk variant (rs7982) in the 5th exon of CLU on alternative splicing by using an integrative approach of brain-tissue-based RNA-Seq and whole genome sequencing data from Accelerating Medicines Partnership-Alzheimer's Disease (AMP-AD). RNA-Seq data were generated from three regions in the temporal lobe of the brain-the temporal cortex, superior temporal gyrus, and parahippocampal gyrus. The rs7982 was significantly associated with intron retention (IR) of the 5th exon of CLU; as the number of alternative alleles (G) increased, the IR rates decreased more significantly in females than in males. Our results suggest a sex-dependent role of rs7982 in AD pathogenesis via splicing regulation.
| S-EPMC7582367 | biostudies-literature
Project description:More than 60% of myeloid dysplasia syndrome (MDS) contains mutations in genes encoding for splicing factors such as SF3B1, U2AF, SRSF2 and ZRSR2. Mutations in SF3B1 are associated with 80% cases of refractory anemia with ring sideroblast (RARS), a subtype of MDS. SF3B1K700E is the most frequently mutated site among mutations on SF3B1. Yet the molecular mechanisms on how mutations of splicing factors lead to defective erythropoiesis are not clear. SF3B1K700E mutant binds to an RNA binding protein, RBM15, stronger than the wild type SF3B1 protein in co-immunoprecipitation assays. In addition, K700E mutant alters the RNA splicing of transcription factors TAL1 and GATA1. Via alternative RNA splicing, a novel short TAL1 transcript variant (TAL1s) is generated. Enhanced interaction between SF3B1 and RBM15 promotes the production of full-length TAL1 (TAL1fl) mRNA, while reduction of RBM15 protein level via PRMT1-mediated degradation pathway changes TAL1s/TAL1fl ratio in favor of TAL1s. TAL1s contains the helix-loop-helix DNA binding domain but not the N terminal region upstream of the DNA binding domain. The TAL1s protein loses its interaction with ETO2, which represses early erythropoiesis. In this vein, overexpression of TAL1s stimulates the transcription of β-hemoglobin in human leukemia K562 cells and promotes erythroid differentiation of human cord blood CD34+ cells cultured in erythropoietin-containing medium. Therefore, mutations of SF3B1 may block erythropoiesis via dysregulation of alternative RNA splicing of transcription factor TAL1, and targeting PRMT1 may alleviate the anemic symptoms in MDS patients.
| S-EPMC5436638 | biostudies-literature
Project description:Mutant p53 (mtp53) promotes chemotherapy resistance through multiple mechanisms, including disabling proapoptotic proteins and regulating gene expression. Comparison of genome wide analysis of mtp53 binding revealed that the ETS-binding site motif (EBS) is prevalent within predicted mtp53-binding sites. We demonstrate that mtp53 regulates gene expression through EBS in promoters and that ETS2 mediates the interaction with this motif. Importantly, we identified TDP2, a 5'-tyrosyl DNA phosphodiesterase involved in the repair of DNA damage caused by etoposide, as a transcriptional target of mtp53. We demonstrate that suppression of TDP2 sensitizes mtp53-expressing cells to etoposide and that mtp53 and TDP2 are frequently overexpressed in human lung cancer; thus, our analysis identifies a potentially "druggable" component of mtp53's gain-of-function activity.
| S-EPMC3337457 | biostudies-literature
Project description:Mutations in splicing factors (SF) are the predominant class of mutations in myelodysplastic syndrome (MDS), but convergent downstream disease drivers remain elusive. To identify common direct targets of missplicing by mutant U2AF1 and SRSF2, we performed RNA sequencing and enhanced version of the cross-linking and immunoprecipitation assay in human hematopoietic stem/progenitor cells derived from isogenic induced pluripotent stem cell (iPSC) models. Integrative analyses of alternative splicing and differential binding converged on a long isoform of GNAS (GNAS-L), promoted by both mutant factors. MDS population genetics, functional and biochemical analyses support that GNAS-L is a driver of MDS and encodes a hyperactive long form of the stimulatory G protein alpha subunit, Gαs-L, that activates ERK/MAPK signaling. SF-mutant MDS cells have activated ERK signaling and consequently are sensitive to MEK inhibitors. Our findings highlight an unexpected and unifying mechanism by which SRSF2 and U2AF1 mutations drive oncogenesis with potential therapeutic implications for MDS and other SF-mutant neoplasms.SignificanceSF mutations are disease-defining in MDS, but their critical effectors remain unknown. We discover the first direct target of convergent missplicing by mutant U2AF1 and SRSF2, a long GNAS isoform, which activates G protein and ERK/MAPK signaling, thereby driving MDS and rendering mutant cells sensitive to MEK inhibition. This article is highlighted in the In This Issue feature, p. 587.
| S-EPMC8904276 | biostudies-literature
Project description:Polycystic ovary syndrome (PCOS) is a common endocrinopathy characterized by hyperandrogenism and metabolic disorders. The excess androgens may be of both ovarian and adrenal origin. PCOS has a strong genetic component, and genome-wide association studies have identified several candidate genes, notably DENND1A, which encodes connecdenn 1, involved in trafficking of endosomes. DENND1A encodes two principal variants, V1 (1009 amino acids) and V2 (559 amino acids). The androgen-producing ovarian theca cells of PCOS women over-express V2. Knockdown of V2 in these cells reduces androgen production, and overexpression of V2 in normal theca cells confers upon them a PCOS phenotype of increased androgen synthesis. We report that human adrenal NCI-H295A cells express V1 and V2 mRNA and that the V2 isoform is produced by exonization of sequences in intron 20, which generates a unique exon 20A, encoding the C-terminus of V2. As in human theca cells from normal women, forced expression of V2 in NCI-H295A cells resulted in increased abundance of CYP17A1 and CYP11A1 mRNAs. We also found genetic variation in the intronic region 330 bp upstream from exon 20A, which could have the potential to drive the selective expression of V2. There was no clear association with these variants with PCOS when we analyzed genomc DNA from normal women and women with PCOS. Using minigene expression vectors in NCI-H295A cells, this variable region did not consistently favor splicing of the V2 transcript. These findings suggest increased V2 expression in PCOS theca cells is not the result of genomic sequence variation in intron 20.
| S-EPMC4983473 | biostudies-literature
Project description:The retinitis pigmentosa GTPase regulator interacting protein 1-like (RPGRIP1L) gene encodes a ciliary protein that is critical for processes related to brain development, including development of left-right asymmetry, sonic hedgehog signaling, and neural tube formation. RPGRIP1L is a risk factor for retinal degeneration, and rare, deleterious variants in the RPGRIP1L gene cause Joubert syndrome and Meckel syndrome, both autosomal recessive disorders. These syndromes are characterized by dysfunctional primary cilia that result in abnormal development - and even lethality in the case of Meckel syndrome. Genetic studies have also implicated RPGRIP1L in psychiatric disorders by suggestive findings from genome-wide association studies and findings from rare-variant exome analyses for bipolar disorder and de novo mutations in autism. In this study we identify a common variant in RPGRIP1L, rs7203525, that influences alternative splicing, increasing the inclusion of exon 20 of RPGRIP1L. We detected this alternative splicing association in human postmortem brain tissue samples and, using a minigene assay combined with in vitro mutagenesis, confirmed that the alternative splicing is attributable to the alleles of this variant. The predominate RPGRIP1L isoform expressed in adult brains does not contain exon 20; thus, a shift to include this exon may impact brain function.
| S-EPMC7206584 | biostudies-literature
Project description:ObjectiveAutosomal-dominant familial Alzheimer disease (AD) is caused by by variants in presenilin 1 (PSEN1), presenilin 2 (PSEN2), and amyloid precursor protein (APP). Previously, we reported a rare PSEN2 frameshift variant in an early-onset AD case (PSEN2 p.K115Efs*11). In this study, we characterize a second family with the same variant and analyze cellular transcripts from both patient fibroblasts and brain lysates.MethodsWe combined genomic, neuropathological, clinical, and molecular techniques to characterize the PSEN2 K115Efs*11 variant in two families.ResultsNeuropathological and clinical evaluation confirmed the AD diagnosis in two individuals carrying the PSEN2 K115Efs*11 variant. A truncated transcript from the variant allele is detectable in patient fibroblasts while levels of wild-type PSEN2 transcript and protein are reduced compared to controls. Functional studies to assess biological consequences of the variant demonstrated that PSEN2 K115Efs*11 fibroblasts secrete less Aβ 1-40 compared to controls, indicating abnormal γ-secretase activity. Analysis of PSEN2 transcript levels in brain tissue revealed alternatively spliced PSEN2 products in patient brain as well as in sporadic AD and age-matched control brain.InterpretationThese data suggest that PSEN2 K115Efs*11 is a likely pathogenic variant associated with AD. We uncovered novel PSEN2 alternative transcripts in addition to previously reported PSEN2 splice isoforms associated with sporadic AD. In the context of a frameshift, these alternative transcripts return to the canonical reading frame with potential to generate deleterious protein products. Our findings suggest novel potential mechanisms by which PSEN variants may influence AD pathogenesis, highlighting the complexity underlying genetic contribution to disease risk.
| S-EPMC6469258 | biostudies-literature