A synonymous UPF3B variant causing a speech disorder implicates NMD as a regulator of neurodevelopmental disorder gene networks

2020 ◽  
Vol 29 (15) ◽  
pp. 2568-2578
Author(s):  
Deepti Domingo ◽  
Urwah Nawaz ◽  
Mark Corbett ◽  
Josh L Espinoza ◽  
Katrina Tatton-Brown ◽  
...  
Keyword(s):  
Cell Lines ◽  
Rna Splicing ◽  
Gene Networks ◽  
Neurodevelopmental Disorder ◽  
Mutant Cell ◽  
Premature Termination Codon ◽  
Loss Of Function ◽  
Altered Expression ◽  
Functional Studies ◽  
Language Disabilities

Abstract Loss-of-function mutations of the X-chromosome gene UPF3B cause male neurodevelopmental disorders (NDDs) via largely unknown mechanisms. We investigated initially by interrogating a novel synonymous UPF3B variant in a male with absent speech. In silico and functional studies using cell lines derived from this individual show altered UPF3B RNA splicing. The resulting mRNA species encodes a frame-shifted protein with a premature termination codon (PTC) predicted to elicit degradation via nonsense-mediated mRNA decay (NMD). UPF3B mRNA was reduced in the cell line, and no UPF3B protein was produced, confirming a loss-of-function allele. UPF3B is itself involved in the NMD mechanism which degrades both PTC-bearing mutant transcripts and also many physiological transcripts. RNAseq analysis showed that ~1.6% of mRNAs exhibited altered expression. These mRNA changes overlapped and correlated with those we identified in additional cell lines obtained from individuals harbouring other UPF3B mutations, permitting us to interrogate pathogenic mechanisms of UPF3B-associated NDDs. We identified 102 genes consistently deregulated across all UPF3B mutant cell lines. Of the 51 upregulated genes, 75% contained an NMD-targeting feature, thus identifying high-confidence direct NMD targets. Intriguingly, 22 of the dysregulated genes encoded known NDD genes, suggesting UPF3B-dependent NMD regulates gene networks critical for cognition and behaviour. Indeed, we show that 78.5% of all NDD genes encode a transcript predicted to be targeted by NMD. These data describe the first synonymous UPF3B mutation in a patient with prominent speech and language disabilities and identify plausible mechanisms of pathology downstream of UPF3B mutations involving the deregulation of NDD-gene networks.

Single-arm study of bimiralisib in head and neck squamous cell carcinoma (HNSCC) patients (pts) harboring NOTCH1 loss of function (LOF) mutations.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. TPS6590-TPS6590
Author(s):  
Faye M. Johnson ◽  
Filip Janku ◽  
J. Jack Lee ◽  
Debora Schmitz ◽  
Henk Streefkerk ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Cell Lines ◽  
Clonal Evolution ◽  
Mutant Cell ◽  
Objective Response ◽  
Site Directed Mutagenesis ◽  
Loss Of Function ◽  
Null Cell ◽  
Second Stage ◽  
Notch1 Mutations

TPS6590 Background: Effective targeted therapies are needed for HNSCC that is lethal despite recent advances with immunotherapy. A major challenge to personalize treatment is that most genomic alterations are in tumor suppressors, including NOTCH1 that is mutated in ~20% of HNSCC. We recently published that HNSCC cell lines harboring NOTCH1 LOF mutations undergo cell death in vivo and in vitro following PI3K inhibition, in contrast to PIK3CA mutant cell lines that merely undergo cell cycle arrest when exposed to the same drugs. Based on these results we initiated a novel genomic biomarker-driven phase II clinical trial treating NOTCH1 mutant HNSCC pts with the dual PI3K/mTOR inhibitor bimiralisib (PQR309). Methods: The primary objective is to determine the objective response rate (ORR) of recurrent/metastatic HNSCC harboring NOTCH1 LOF mutations to bimiralisib. Pts who have already received standard platinum chemotherapy and immunotherapy will receive bimiralisib orally twice per wk unless progression or intolerable toxicity occurs. Tumors will be evaluated using RECIST q 6 wks. A Simon’s optimal two-stage design is used. To have 80% power to detect an ORR of 30%, (one-sided α = 0.05, β = 0.20) 10 pts will be enrolled in the first stage. If ≤1 pts respond, the trial will be closed for futility. If ≥2 pts have an OR, the study will enroll an additional 19 pts in the second stage. The null hypothesis (ORR ≤ 10%) will be rejected if ≥ 6 in 29 pts have an OR. Seven pts have enrolled. The algorithm for determining NOTCH1 mutation function is based on the patterns of mutations in HNSCC vs. leukemia where mutations are activating. It may be difficult to determine whether NOTCH1 mutations are homo- or heterozygous due to normal cell contamination. Therefore, levels of activated NOTCH1 in pretreatment tumors may be assessed by IHC with an antibody against activated NOTCH1 (NICD). In parallel with the trial, to further confirm NOTCH1 LOF, we can use site-directed mutagenesis to re-create NOTCH1 mutations from trial pts that will then be introduced into NOTCH1-null cell lines to assay for NICD and growth inhibition with culture on NOTCH1 ligand. All pts will have serial collection of blood for pharmacokinetics and for ctDNA to examine clonal evolution associated with acquired resistance. Samples with high NOTCH1 mutation ctDNA VAF will be analyzed by WES and compared with pretreatment tissue. In the second stage, IHC and WES may be performed on pre- and post- treatment (day 15 and progression) tissue to examine pharmacodynamics and mechanisms of resistance. Clinical trial information: NCT03740100 .

scholarly journals Brain and Breast Cancer Cells with PTEN Loss of Function Reveal Enhanced Durotaxis and RHOB Dependent Amoeboid Migration Utilizing 3D Scaffolds and Aligned Microfiber Tracts

Cancers ◽  
2021 ◽  
Vol 13 (20) ◽  
pp. 5144
Author(s):  
Annalena Wieland ◽  
Pamela L. Strissel ◽  
Hannah Schorle ◽  
Ezgi Bakirci ◽  
Dieter Janzen ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Tumor Cells ◽  
Cell Lines ◽  
Essential Role ◽  
Brain Structures ◽  
Loss Of Function ◽  
Pten Loss ◽  
Tumor Cell Migration ◽  
Functional Studies ◽  
Tnbc Cell

Background: Glioblastoma multiforme (GBM) and metastatic triple-negative breast cancer (TNBC) with PTEN mutations often lead to brain dissemination with poor patient outcome, thus new therapeutic targets are needed. To understand signaling, controlling the dynamics and mechanics of brain tumor cell migration, we implemented GBM and TNBC cell lines and designed 3D aligned microfibers and scaffolds mimicking brain structures. Methods: 3D microfibers and scaffolds were printed using melt electrowriting. GBM and TNBC cell lines with opposing PTEN genotypes were analyzed with RHO-ROCK-PTEN inhibitors and PTEN rescue using live-cell imaging. RNA-sequencing and qPCR of tumor cells in 3D with microfibers were performed, while scanning electron microscopy and confocal microscopy addressed cell morphology. Results: In contrast to the PTEN wildtype, GBM and TNBC cells with PTEN loss of function yielded enhanced durotaxis, topotaxis, adhesion, amoeboid migration on 3D microfibers and significant high RHOB expression. Functional studies concerning RHOB-ROCK-PTEN signaling confirmed the essential role for the above cellular processes. Conclusions: This study demonstrates a significant role of the PTEN genotype and RHOB expression for durotaxis, adhesion and migration dependent on 3D. GBM and TNBC cells with PTEN loss of function have an affinity for stiff brain structures promoting metastasis. 3D microfibers represent an important tool to model brain metastasizing tumor cells, where RHO-inhibitors could play an essential role for improved therapy.

scholarly journals Genome-wide silencing screen in mesothelioma cells reveals that loss of function of BAP1 induces chemoresistance to ribonucleotide reductase inhibition: implication for therapy

2018 ◽  
Author(s):  
Agata Okonska ◽  
Saskja Bühler ◽  
Vasundhara Rao ◽  
Manuel Ronner ◽  
Maxime Blijlevens ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Ribonucleotide Reductase ◽  
Genetically Engineered ◽  
Whole Genome ◽  
Loss Of Function ◽  
Functional Studies ◽  
Genome Wide ◽  
Sirna Screens ◽  
Gemcitabine Treatment

ABSTRACTIntroductionLoss of function of BRCA1 associated protein 1 (BAP1) is observed in about 50% of malignant pleural mesothelioma (MPM) cases. The aim of this study was to investigate whether this aspect could be exploited for targeted therapy.MethodsA genetically engineered model was established expressing either functional or nonfunctional BAP1 and whole-genome siRNA screens were performed assessing impaired survival between the two cell lines. Cytotoxity induced by gemcitabine and hydroxyurea were assessed in a panel of BAP1-WT and BAP1-mut/del cell lines. Functional studies were carried out in BAP1 mut/del cell line reconstituted with BAP1 WT or BAP1 C91A (catalytically dead mutant) and in BAP1 WT cell line upon siRNA-mediated knock-down of BAP1.ResultsThe whole-genome siRNA screen unexpectedly revealed 11 hits (FDR<0.05) more cytotoxic for BAP1-proficient cells. Two actionable targets, RRM1 and RRM2, were validated and their inhibition mediated by gemcitabine or hydroxyurea respectively, was more cytotoxic in BAP1-proficient cell lines. Upregulation of RRM2 upon gemcitabine and hydroxyurea was more profound in BAP1 mut/del cell lines. Increased lethality mediated by gemcitabine and hydroxyurea was observed in NCI-H2452 cells reconstituted with BAP1 WT but not with C91A mutant and upregulation of RRM2 in NCI-H2452-BAP1 WT spheroids was modest compared to control or C91A mutant. Finally, the opposite was observed after BAP1 knockdown in BAP1-proficient SPC111 cell line.ConclusionWe found that BAP1 is involved in the regulation of RRM2 levels during replication stress. These observations reveal a potential therapeutic approach where MPM patients to be stratified depending on BAP status for gemcitabine treatment.

scholarly journals Novel Loss-of-Function Mutations in COCH Cause Autosomal Recessive Nonsyndromic Deafness

2020 ◽  
Author(s):  
Kevin T Booth ◽  
Amama Ghaffar ◽  
Muhammad Rashid ◽  
Luke T Hovey ◽  
Mureed Hussain ◽  
...  
Keyword(s):  
Hearing Loss ◽  
Rna Splicing ◽  
Autosomal Recessive ◽  
Dominant Negative ◽  
Nonsyndromic Hearing Loss ◽  
Loss Of Function ◽  
Functional Studies ◽  
Pathogenic Variants ◽  
Exon Splicing ◽  
Coding Variants

AbstractCOCH is the most abundantly expressed gene in the cochlea. Unsurprisingly, mutations in COCH underly deafness in mice and humans. Two forms of deafness are linked to mutations in COCH, the well-established autosomal dominant nonsyndromic hearing loss, with or without vestibular dysfunction (DFNA9) via a gain-of-function/dominant-negative mechanism, and more recently autosomal recessive nonsyndromic hearing loss (DFNB110) via nonsense variants. Using a combination of targeted gene panels, exome sequencing and functional studies, we identified four novel pathogenic variants (two nonsense variants, one missense and one inframe deletion) in COCH as the cause of autosomal recessive hearing loss in a multi-ethnic cohort. To investigate whether the non-truncating variants exert their effect via a loss-of-function mechanism, we used mini-gene splicing assays. Our data showed both the missense and inframe deletion variants altered RNA-splicing by creating an exon splicing silencer and abolishing an exon splicing enhancer, respectively. Both variants create frameshifts and are predicted to result in a null allele. This study confirms the involvement of loss-of-function mutations in COCH in autosomal recessive nonsyndromic hearing loss, expands the mutational landscape of DFNB110 to include coding variants that alter RNA-splicing, and highlights the need to investigate the effect of coding variants on RNA-splicing.

scholarly journals Deleting a UBE3A substrate rescues impaired hippocampal physiology and learning in Angelman syndrome mice

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Gabrielle L. Sell ◽  
Wendy Xin ◽  
Emily K. Cook ◽  
Mark A. Zbinden ◽  
Thomas B. Schaffer ◽  
...  
Keyword(s):  
Angelman Syndrome ◽  
Developmental Disorders ◽  
Motor Coordination ◽  
Synapse Formation ◽  
Neurodevelopmental Disorder ◽  
Autism Spectrum ◽  
Loss Of Function ◽  
Behavioral Deficits ◽  
Altered Expression ◽  
Exciting Potential

AbstractIn humans, loss-of-function mutations in the UBE3A gene lead to the neurodevelopmental disorder Angelman syndrome (AS). AS patients have severe impairments in speech, learning and memory, and motor coordination, for which there is currently no treatment. In addition, UBE3A is duplicated in > 1–2% of patients with autism spectrum disorders—a further indication of the significant role it plays in brain development. Altered expression of UBE3A, an E3 ubiquitin ligase, is hypothesized to lead to impaired levels of its target proteins, but identifying the contribution of individual UBE3A targets to UBE3A-dependent deficits remains of critical importance. Ephexin5 is a putative UBE3A substrate that has restricted expression early in development, regulates synapse formation during hippocampal development, and is abnormally elevated in AS mice, modeled by maternally-derived Ube3a gene deletion. Here, we report that Ephexin5 can be directly ubiquitylated by UBE3A. Furthermore, removing Ephexin5 from AS mice specifically rescued hippocampus-dependent behaviors, CA1 physiology, and deficits in dendritic spine number. Our findings identify Ephexin5 as a key driver of hippocampal dysfunction and related behavioral deficits in AS mouse models. These results demonstrate the exciting potential of targeting Ephexin5, and possibly other UBE3A substrates, to improve symptoms of AS and other UBE3A-related developmental disorders.

scholarly journals Functional Genomic Landscape of Genes with Recurrent Mutations in Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 189-189 ◽  
Author(s):  
Ricardo De Matos Simoes ◽  
Ryosuke Shirasaki ◽  
Huihui Tang ◽  
Michal Sheffer ◽  
Olga Dashevsky ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Tumor Cell ◽  
Cell Survival ◽  
Cell Lines ◽  
Research Funding ◽  
Functional Genomic ◽  
Loss Of Function ◽  
Functional Studies ◽  
Tumor Cell Survival ◽  
Genome Scale

Abstract During the last two decades, cell lines and patient-derived samples from multiple myeloma (MM) have been extensively profiled for alterations in their genome with the anticipation that those genes with the most recurrent lesions could represent attractive novel therapeutic targets or markers for aggressive disease. Yet for many of these genes, their functional significance for MM cells has not been formally evaluated. With the advent of new CRISPR/Cas9-based functional genomics platforms, it is possible to generate in genome- or subgenome-scale direct quantitative information on the impact that perturbation of these genes exerts on tumor cell survival, proliferation or other phenotypes. We therefore examined the landscape of our CRISPR-based functional genomic data for these recurrently dysregulated genes We specifically curated information from the MMRF CoMMpass study and multiple other publicly available studies, to identify genes which are recurrently identified to harbor nonsynonymous mutation (SNV or indel), DNA copy number loss or gain, or participation in chromosomal translocations. We then examined the patterns of results for these genes in our genome scale CRISPR-based gene-editing studies for loss-of-function in n=18 MM cell lines. We identified a subset of genes (e.g. FAM46C, CDKN2C, RASA2) which are considered targets for recurrent loss-of-function events and indeed exhibit, for large fractions of the cell lines tested enrichment, of their sgRNAs in CRISPR knock-out studies, consistent with a role of these genes as suppressors of tumor cell survival or proliferation. CRISPR KO of TP53 leads to increased survival/proliferation of only a small minority (2/18 of cell lines tested thus far), which reflects the fact that the overwhelming majority of MM cell lines already harbor LOF events for this gene. Interestingly, a substantial number of genes which have been considered to harbor recurrent LOF events in MM patient samples (e.g. NF1, NF2, CYLD) do not exhibit sgRNA enrichment in CRISPR KO screens in the MM cell lines tested so far. In addition, several other recurrently mutated genes for which their loss- or gain-of-function status had not been previously evaluated with extensive functional studies in MM (e.g. SP140, LTB, EGR1, ATM, PARK2, PRKD2, RAPGEF5, DOCK5, TGDS, TNFAIP8) exhibit in the majority of cell lines tested in in CRISPR knockout studies no significant enrichment or depletion of their sgRNAs. In contrast, PTPN11, CREBBP, EP300, KMT2B, KMT2C, SETD2, SF3B1 and UBR5, are notable examples of recurrently mutated genes which represent dependencies for large fractions of MM cell lines in vitro. These results highlight the value of interpreting results from next generation sequencing studies in the context of information provided by the genome scale by use of functional genomic characterization of available cell line models. We envision that, similar sub-genome scale assays were performed at the level of patient derived samples will also provide direct information about the relevance of some of these genes. In addition, functional studies conducted with context of tumor-microenvironemtn compartment interactions and tumor interface will be needed to evaluate several genes identified in the study. Disclosures Licht: Celgene: Research Funding. Mitsiades:Takeda: Other: employment of a relative; Janssen/ Johnson & Johnson: Research Funding; Abbvie: Research Funding; EMD Serono: Research Funding; TEVA: Research Funding.

scholarly journals Dystrophins, Utrophins, and Associated Scaffolding Complexes: Role in Mammalian Brain and Implications for Therapeutic Strategies

2010 ◽  
Vol 2010 ◽  
pp. 1-19 ◽  
Author(s):  
Caroline Perronnet ◽  
Cyrille Vaillend
Keyword(s):  
Dystrophin Gene ◽  
Experimental Manipulation ◽  
Therapeutic Strategies ◽  
Mammalian Brain ◽  
Loss Of Function ◽  
Altered Expression ◽  
Functional Studies ◽  
Cellular Expression ◽  
Structural And Functional Properties ◽  
Brain Expression

Two decades of molecular, cellular, and functional studies considerably increased our understanding of dystrophins function and unveiled the complex etiology of the cognitive deficits in Duchenne muscular dystrophy (DMD), which involves altered expression of several dystrophin-gene products in brain. Dystrophins are normally part of critical cytoskeleton-associated membrane-bound molecular scaffolds involved in the clustering of receptors, ion channels, and signaling proteins that contribute to synapse physiology and blood-brain barrier function. The utrophin gene also drives brain expression of several paralogs proteins, which cellular expression and biological roles remain to be elucidated. Here we review the structural and functional properties of dystrophins and utrophins in brain, the consequences of dystrophins loss-of-function as revealed by numerous studies in mouse models of DMD, and we discuss future challenges and putative therapeutic strategies that may compensate for the cognitive impairment in DMD based on experimental manipulation of dystrophins and/or utrophins brain expression.

scholarly journals Identification of Gene Networks Associated with the Anti-Leukemic Effect of Anti-Inflammatory Drugs on Acute Myeloid Leukemia Cell Lines

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4343-4343
Author(s):  
Victória Tomaz ◽  
Karina Griesi-Oliveira ◽  
Renato D Puga ◽  
Fabio Pires de Souza Santos ◽  
Nelson Hamerschlak ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
Board Of Directors ◽  
Cell Lines ◽  
Rna Splicing ◽  
Gene Networks ◽  
Functional Enrichment ◽  
Advisory Committees ◽  
Cycle Arrest ◽  
Anti Inflammatory

Abstract Introduction Despite recent advances in therapy, acute myeloid leukemia (AML) remain a medical challenge with high morbidity and mortality rates. For most patients, allogeneic hematopoietic stem cell transplantation remain the only curative option, but due to the advanced age at diagnosis, a significant proportion of patients are not elegible to this form of therapy. Nevertheless, novel therapies are warranted. There is preclinical evidence that anti-inflammatory compounds, such as COX-2 inhibitors and steroids, may have anti-neoplastic activity in different tumor types, including AML; nevertheles the mechanisms associated with its anti-neoplastic activity are not clear. Therefore, the aim of this work was to evaluate the anti-leukemic effect of the anti-inflammatory compounds nimesulide and prednisolone in AML cell lines and to identify genes and molecular pathways associated with cytotoxicity through transcriptome analysis. Methods The leukemic cell lines HL-60, THP-1, OCI-AML2 and OCI-AML3 were treated with nimesulide and prednisolone at 100 µM alone and in combination and with cytarabine at 2.5 µM. Twenty four hours after treatment , we measured the amount of cell death using Annexin V Apoptosis Detection Kit FITC (ThermoFisher) and the cell cycle was analized after fixing the cells with 70% alcohol and incubation with propidium iodide (1mg/ml) and RNAse (10mg/ml). In another experiment, we harvested the cells after 4 hours of treatment for transcriptome analysis. RNA was extracted from control (DMSO) and treatment groups (1 - nimesulide, 2 - prednisolone , 3 - nimesulide and prednisolone) with RNeasy Mini Kit (Qiagen). The Illumina® NEBNext® Ultra II Directional RNA Library Prep Kit was used for library preparation, following the manufacturer protocol using Poly(A) mRNA Magnetic Isolation Module. Equimolar amount of libraries was sequenced using an Illumina NextSeq 500, following the manufacturer's instructions, on the Oklahoma Medical Research Foundation Genomics Core (USA). The sequences obtained with the RNA-Seq technique were aligned in the human genome of reference GRCh37.75 by the software Spliced Transcripts Alignment to a Reference (STAR) v2.5 and to obtain normalized counts in FPKM, the software Expectation-Maximization (RSEM) v1.3.0 was used. To identify network of genes correlated with the treatment (modules), we used the Weighted Correlation Network Analysis (WGCNA). Functional enrichment analysis of the WGCNA differentially expressed modules was performed using the Integrated Annotation, Visualization and Discovery Database (DAVID) v6.8 in order to correlate with biological processes. Results In the cell cycle analysis, we observed a significant increase (p &lt; 0.05) in the sub-G0 phase (cell death) after treatment with nimesulide alone, and in combination with prednisolone (figure 1). No effect was observed in the prednisolone only group. The cell cycle effect induced by nimesulide on HL-60 and OCI-AML2 was similar to the induced by cytarabine, a standard chemotherapy agent for AML that in known to induce arrest in the S phase. In addition, the cell line arrest in THP-1 was greater with nimesulide than with cytarabine, while OCI-AML3 was less sensitive to both nimesulide and cytarabine. Regarding cell death mechanism, treatment with nimesulide induced predominantly an increase in late apoptosis that was potentiated after combined treatment with nimesulide and cytarabine (figure 2). After the demonstration of cell cycle arrest and apoptosis induction after treatment with nimesulide, we performed whole transcriptome sequencing followed by WGCNA analysis. We have identified gene modules that were significantly correlated with anti-inflammatory treatments, being 1 module down-regulated (lightyellow with p = 0.00052) and 2 modules up-regulated (lightcyan with p = 0 .00025 and tan with p = 0.000038). Analysis of functional enrichment using DAVID showed up-regulation of gene networks associated with apoptotic processes and autophagy and down- regulation of gene networks associated with cell cycle and RNA splicing pathways Conclusions The COX-2 inhibitor nimesulide caused cell cycle arrest and apoptosis in AML cell lines and potentiated the cytotoxic effects of cytarabine. This treatment was associated with up- regulation of autophagy and apoptosis and down-regulation of cell cycle and RNA splicing gene networks. Figure 1 Figure 1. Disclosures Santos: Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees. Campregher: Astellas: Consultancy.

scholarly journals Deleting a UBE3A substrate rescues impaired hippocampal physiology and learning in Angelman syndrome mice

2019 ◽  
Author(s):  
Gabrielle L. Sell ◽  
Wendy Xin ◽  
Emily K. Cook ◽  
Mark A. Zbinden ◽  
Thomas B. Schaffer ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Angelman Syndrome ◽  
Developmental Disorders ◽  
Motor Coordination ◽  
Synapse Formation ◽  
Neurodevelopmental Disorder ◽  
Autism Spectrum ◽  
Loss Of Function ◽  
Altered Expression ◽  
Exciting Potential

ABSTRACTIn humans, loss-of-function mutations in the UBE3A gene lead to the neurodevelopmental disorder Angelman syndrome (AS). AS patients have severe impairments in speech, learning and memory, and motor coordination, for which there is currently no treatment. In addition, UBE3A is duplicated in >1-2% of patients with autism spectrum disorders – a further indication of the significant role it plays in brain development. Altered expression of UBE3A, an E3 ubiquitin ligase, is hypothesized to lead to impaired levels of its target proteins, but identifying the contribution of individual UBE3A targets to UBE3A-dependent deficits remains of critical importance. Ephexin5 is a putative UBE3A substrate that has restricted expression early in development, regulates synapse formation during hippocampal development, and is abnormally elevated in AS mice, modeled by maternally-derived Ube3a gene deletion. Here, we report that Ephexin5 is a direct substrate of UBE3A ubiquitin ligase activity. Furthermore, removing Ephexin5 from AS mice specifically rescued hippocampus-dependent behaviors, CA1 physiology, and deficits in dendritic spine number. Our findings identify Ephexin5 as a key driver of hippocampal dysfunction and related behavioral deficits in AS mouse models. These results demonstrate the exciting potential of targeting Ephexin5, and possibly other UBE3A substrates, to improve symptoms of AS and other UBE3A-related developmental disorders.

scholarly journals U2AF1 Mutations Cause Allele-Specific Alterations in 3' Splice Site Recognition in Myeloid Malignancies

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1889-1889 ◽  
Author(s):  
Janine O. Ilagan ◽  
Aravind Ramakrishnan ◽  
Brian Hayes ◽  
Michele E. Murphy ◽  
Ahmad S. Zebari ◽  
...  
Keyword(s):  
Cell Lines ◽  
Splice Site ◽  
Rna Splicing ◽  
K562 Cells ◽  
Site Preference ◽  
Loss Of Function ◽  
Myeloid Malignancies ◽  
Before And After ◽  
Allele Specific

Abstract Mutations affecting the spliceosomal protein U2AF1 are among the most common mutations observed in patients with MDS and related disorders. However, it is unclear how these mutations affect the normal RNA splicing process, and how the resulting changes in splicing contribute to myeloid dysplasia. Here, we combined the strengths of data from primary AML patient samples with the controlled context of isogenic cell lines. We generated K562 erythroleukemic cell lines stably expressing each of the four common U2AF1 mutations (S34F, S34Y, Q157P, and Q157R). We compared expression of each of these mutant alleles with knock down of endogenous U2AF1 to compare the relative consequences of U2AF1 mutations versus loss of function. We first sought to identify changes in splicing driven by U2AF1 mutations that contribute to myeloid dysplasia. We compared the splicing of ~125,000 annotated alternative splicing events and ~160,000 constitutively spliced junctions between AML samples with or without mutations (TCGA cohort), as well as our isogenic K562 cell lines stably expressing either mutant (S34F, S34Y, Q157P, and Q157R) or wild-type (WT) alleles of U2AF1. Unsupervised cluster analysis revealed that S34F/Y versus Q157P/R samples clustered together in both the AML data and our cell lines, suggesting that U2AF1 mutations affecting different residues of the protein have different molecular consequences. Intersecting the AML and K562 data, we identified >300 splicing events that were consistently differentially spliced in association with S34 mutations, and a similar number for Q157 mutations. Many of these splicing events affected biological pathways that have been implicated in myeloid malignancies, including DNA methylation (DNMT3B), X chromosome inactivation (H2AFY), the DNA damage response (ATR, FANCA), and apoptosis (CASP8). For example, two exons of DNMT3B are differentially spliced in both AML samples and our K562 cells (Figure A), including an exon lying within the methyltransferase domain. We next identified mechanistic changes in the splicing process caused by U2AF1 mutations. U2AF1 binds the intron-exon boundary by sequence-specifically recognizing the AG dinucleotide and flanking sequence positions that define the 3' splice site. Comparing AML samples and K562 cells with and without U2AF1 mutations, we found that S34 and Q157 mutations give rise to specific and distinct alterations in 3' splice site preference. S34 mutations alter the consensus nucleotide immediately before the AG dinucleotide, while Q157 mutations alter the consensus nucleotide immediately after the AG (Figure B). We observed highly similar allele-specific alterations in 3' splice site preference in every AML patient with a U2AF1 mutation, as well as all K562 cell lines expressing a U2AF1 mutant allele. In contrast, knock down of endogenous U2AF1 caused no alterations in the consensus sequence at those positions, indicating that U2AF1 mutations do not cause loss of function at the level of RNA splicing. To confirm that the nucleotides immediately before and after the AG determine whether a splice site responds to U2AF1 mutations, we created minigenes of cassette exons within the ATR and EPB49 genes. We found that response to U2AF1 S34 and Q157 mutations requires the endogenous nucleotides immediately before and after the AG, as predicted by our genomics analysis, and that mutating these positions abolishes response to U2AF1 mutations. Finally, we recapitulated the RNA splicing process in vitro using nuclear extract from blood cells expressing either wild-type or mutant U2AF1 to show that identical changes in splice site preference occur in a controlled in vitro context (Figure C). Together, our data show that U2AF1 mutations cause allele-specific alterations in normal 3' splice site recognition in patients, in isogenic cell lines, and in vitro. These alterations in splice site preference give rise to mis-splicing that affects many genes previously implicated in myeloid malignancies. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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