Project description:In order to better understand chondrodysplasia disease mechanisms, we induced hypertrophic chondrocytes from chondrodysplasia-specific iPSCs and analyzed their gene expression profile. This dataset includes the expression data obtained from iPSC-derived cartilage pellets on day 56 of hypertrophic induction. Three COL10A1 mutants and two MATN3 mutants were compared to their isogenic controls to determine the effects of each mutation on the unfolded protein response.
Project description:V156D matn1 mutation (homologous to the V194D matn3 mutation causing Multiple Epiphyseal Dysplasia (MED)) was introduced by gene targetting in order to evaluate the potential of matrilin-1 to be a novel chondrodysplasia based on domain homology and aminoacid conservation. We used microarrays to analyse the differential expression of genes between wild type and mutant animals at day 5 (postnatal)
Project description:Many cancers carry recurrent change-of-function mutations in RNA splicing factor genes, which induce sequence-specific changes in RNA splicing. Here, we describe a method to harness this change in RNA splicing activity to drive splicing factor mutation-dependent gene expression in cancers and selectively eliminate these tumors. We engineered synthetic introns which were efficiently spliced in leukemia and breast epithelial cells bearing the most common SF3B1 mutations, but unspliced in wild-type cells—and vice versa—to yield mutation-dependent protein production. A massively parallel screen of 8,881 distinct introns delineated ideal intronic size, mapped essential sequence elements, and revealed the basis of mutation-dependent splicing. Synthetic introns enabled mutation-dependent expression of herpes simplex virus thymidine kinase and subsequent ganciclovir-mediated elimination of leukemia and breast epithelial cells bearing SF3B1 mutations, while leaving wild-type cells unaffected. This approach significantly decreased the growth of otherwise lethal leukemia xenografts and correspondingly improved host survival. The modular, compact, and specific nature of synthetic introns thereby provide a means to exploit cancer-specific changes in RNA splicing for genotype-dependent gene expression and gene therapy.
Project description:Achondroplasia (ACH), the most common form of human dwarfism is caused by a mutation in the Fibroblast Growth Factor Receptor 3 (FGFR3) gene, resulting in constitutive activation of the receptor. In order to gain insight into molecular mechanisms involved in the physiopathology, a conditional mouse model carrying the Y367C mutation corresponding to the human Y373C TDI mutation was produced (fgfr3neoY367C/+). Crossing these mice with CMV-CRE mice produces dwarf fgfr3Y367C/+ and fgfr3+/+ controls littermates. Dwarf fgfr3Y367C/+ mice exhibited skeletal dysplasia at birth, the phenotype becoming progressively more pronounced as the mice got older, with reduced length of long bone, narrow trunk, short ribs and macrocephaly. Histological examination of epiphyseal growth plates showed a disorganized growth plate, with reduced size of the hypertrophic and proliferative zone and an accelerated secondary ossification center formation (full phenotype description in Pannier S et al, Activating Fgfr3 Y367C mutation causes hearing loss and inner ear defect in a mouse model of chondrodysplasia. Biochimica et biophysica acta 2009; Pannier S et al, Delayed bone age due to a dual effect of FGFR3 mutation in Achondroplasia. Bone 2010; Jonquoy A et al, A novel tyrosine kinase inhibitor restores chondrocyte differentiation and promotes bone growth in a gain-of-function Fgfr3 mouse model. Human molecular genetics 2012; Martin L et al, Constitutively-active FGFR3 disrupts primary cilium length and IFT20 trafficking in various chondrocyte models of achondroplasia. Human molecular genetics 2018).
Project description:How genetic defects trigger late-onset disease is important for understanding disease progression and therapeutic development. Fuchs’ endothelial corneal dystrophy (FECD) is an RNA-mediated disease caused by a trinucleotide CUG expansion in an intron within the TCF4 gene. The mutant intronic CUG RNA is present at 1-2 copies per cell, posing a challenge to understand how a rare RNA can cause disease. Late-onset FECD is a uniquely advantageous model for studying how RNA triggers disease because; 1) Affected tissue is routinely removed during surgery; 2) The expanded CUG mutation is one of the most prevalent disease-causing mutations, making it possible to obtain pre-symptomatic tissue from eye bank donors to probe how gene expression changes precede disease; and 3) The affected tissue is a homogeneous single cell monolayer, facilitating accurate transcriptome analysis. Here we use RNA sequencing to compare tissue from individuals who are pre-symptomatic (Pre_S) to tissue from patients with late stage FECD (FECD_REP). The abundance of mutant repeat intronic RNA in Pre_S and FECD_REP tissue is elevated due to increased half-life in a corneal cell-specific manner. In Pre_S tissue, changes in splicing and extracellular matrix gene expression foreshadow the changes observed in advanced disease and predict the activation of the fibrosis pathway and immune system seen in late-stage patients. The absolute efficiency of splicing changes are similar in presymptomatic and late stage tissue. Our data identify gene candidates for early drivers of disease and biomarkers that may represent diagnostic and therapeutic targets for FECD. We conclude that changes in alternative splicing and gene expression are observable decades prior to the diagnosis of late-onset trinucleotide repeat disease.
Project description:Purpose: DMD pathogenic variants for Duchenne and Becker muscular dystrophy are detectable with high sensitivity by standard clinical exome analyses of genomic DNA. However, up to 7% of DMD mutations are deep intronic and analysis of muscle-derived RNA is an important diagnostic step for patients who have negative genomic testing but abnormal dystrophin expression in muscle. In this study, muscle biopsies were evaluated in 19 patients with clinical features of a dystrophinopathy, but negative clinical DMD mutation analysis. Methods: Reverse transcription PCR (RT-PCR) or high-throughput RNA sequencing (RNA-Seq) methods identified 19 mutations with one of three pathogenic pseudoexon types: deep intronic point mutations, deletions or insertions, and translocations. Results: In association with point mutations creating intronic splice acceptor sites, we observed the first examples of DMD pseudo 3’-terminal exon mutations causing high efficiency transcription termination within introns. This connection between splicing and premature transcription termination is reminiscent of U1 snRNP-mediating telescripting in sustaining RNA polymerase II elongation across large genes, such as DMD. Conclusions: We propose a novel classification of three distinct types of mutations identifiable by muscle RNA analysis, each of which differ in potential treatment approaches. Recognition and appropriate characterization may lead to therapies directed toward full-length dystrophin expression for some patients.
Project description:RNA splicing plays a critical role in post-transcriptional gene regulation. Exponential expansion of intron length poses a challenge for accurate splicing. Little is known about how cells prevent inadvertent and often deleterious expression of intronic elements due to cryptic splicing. In this study, we identify hnRNPM as an essential RNA binding protein that suppresses cryptic splicing through binding to deep introns, preserving transcriptome integrity. Long interspersed nuclear elements (LINEs) harbor large amounts of pseudo splice sites in introns. hnRNPM preferentially binds at intronic LINEs and represses LINE-containing pseudo splice site usage for cryptic splicing. Remarkably, a subgroup of the cryptic exons can form long dsRNAs through base-pairing of inverted Alu transposable elements scattered in between LINEs and trigger interferon immune response, a well-known antiviral defense mechanism. Notably, these interferon-associated pathways are found to be upregulated in hnRNPM-deficient tumors, which also exhibit elevated immune cell infiltration. These findings unveil hnRNPM as a guardian of transcriptome integrity. Targeting hnRNPM in tumors may be used to trigger an inflammatory immune response thereby boosting cancer surveillance.
Project description:RNA splicing plays a critical role in post-transcriptional gene regulation. Exponential expansion of intron length poses a challenge for accurate splicing. Little is known about how cells prevent inadvertent and often deleterious expression of intronic elements due to cryptic splicing. In this study, we identify hnRNPM as an essential RNA binding protein that suppresses cryptic splicing through binding to deep introns, preserving transcriptome integrity. Long interspersed nuclear elements (LINEs) harbor large amounts of pseudo splice sites in introns. hnRNPM preferentially binds at intronic LINEs and represses LINE-containing pseudo splice site usage for cryptic splicing. Remarkably, a subgroup of the cryptic exons can form long dsRNAs through base-pairing of inverted Alu transposable elements scattered in between LINEs and trigger interferon immune response, a well-known antiviral defense mechanism. Notably, these interferon-associated pathways are found to be upregulated in hnRNPM-deficient tumors, which also exhibit elevated immune cell infiltration. These findings unveil hnRNPM as a guardian of transcriptome integrity. Targeting hnRNPM in tumors may be used to trigger an inflammatory immune response thereby boosting cancer surveillance.
Project description:Pre-mRNA splicing is a key control point in human gene expression. Disturbances in splicing due to mutation or aberrant splicing regulatory networks lead to dysregulated protein expression and contribute to a substantial fraction of human disease. Several classes of active and selective splicing modulator compounds (SMCs) have been recently identified and establish that pre-mRNA splicing represents a viable target for therapy. This also raises the intriguing possibility that SMCs may have broad capabilities to ameliorate aberrant splicing across multiple human disorders. We describe herein the identification of BPN-15477, a novel SMC that restores correct splicing of exon 20 in the Elongator complex protein 1 (ELP1) gene carrying the major IVS20+6T>C mutation responsible for familial dysautonomia. Given that BPN-15477 corrects splicing and increases full-length ELP1 protein in vivo, we developed a machine learning approach to evaluate the therapeutic potential of BPN-15477 to correct splicing in other human genetic diseases. Using transcriptome sequencing from compound-treated fibroblast cells, we identified treatment responsive sequence signatures, the majority of which center at the 5' splice site of exons whose inclusion or exclusion is modulated by SMC treatment. We then leveraged this model to identify 155 human disease genes that harbor ClinVar mutations predicted to alter pre-mRNA splicing as potential targets for BPN-15477 treatment. Using in vitro splicing assays, we validated representative predictions by demonstrating successful correction of splicing defects caused by mutations in the genes responsible for cystic fibrosis (CFTR), cholesterol ester storage disease (LIPA), Lynch syndrome (MLH1) and familial frontotemporal dementia (MAPT). Importantly, we also validated these predictions in two disease relevant cellular models for LIPA and CFTR, confirming that treatment increases functional protein and confirming the clinical potential for our model predictions. Our study shows that deep learning techniques can identify a complex set of sequence signatures and predict response to pharmacological modulation, strongly supporting the use of in silico approaches to expand the therapeutic potential of drugs that modulate splicing.
Project description:Background: The presence of nuclear mitochondrial DNA (numtDNA) has been reported within several nuclear genomes. Next to mitochondrial protein coding genes, numtDNA sequences also encode for mitochondrial tRNA genes. However, the biological roles of numtDNA, remain elusive. Results: Employing in silico analysis we identify 281 mitochondrial tRNA homologs in the human genome, which we term nimtRNAs (nuclear intronic mitochondrial-derived tRNAs), being contained within introns of 76 nuclear host genes. Despite base changes in nimtRNAs when compared to their mtRNA homologs, a canonical tRNA cloverleaf structure is maintained. To address potential functions of intronic nimtRNAs, we insert them into introns of constitutive and alternative splicing reporters and demonstrate that nimtRNAs promote pre-mRNA splicing, dependent on number and positioning of nimtRNA genes and splice site recognition efficiency. A mutational analysis reveals that the nimtRNA cloverleaf structure is required for the observed splicing increase. Utilizing a CRISPR/Cas9 approach we show that a partial deletion of a single endogenous nimtRNALys within intron 28 of the PPFIBP1 gene decreases inclusion of the downstream located exon 29 of the PPFIBP1 mRNA. By employing a pull-down approach followed by mass spectrometry, a 3’-splice site associated protein network is identified, including KHDRBS1, which we show directly interacts with nimtRNATyr by an electrophoretic mobility shift assay. Conclusions: We propose that nimtRNAs, along with associated protein factors, can act as a novel class of intronic splicing regulatory elements in the human genome by participating in the regulation of splicing.