EXOSC3 Research Articles

Identification of RNA Exosome Cofactors in Neuronal Cells to Probe Disease Mechanism

The RNA exosome, a 10‐subunit complex that mediates both RNA processing and degradation, plays a critical role in defining cellular expression profiles. This complex is ubiquitously expressed, essential, and critical for fundamental cellular functions, such as ribosomal RNA processing. Recent studies have linked mutations in genes encoding multiple subunits of the complex to tissue‐specific human disease. For example, missense mutations in the human EXOSC3 gene, which encodes an RNA exosome subunit, cause Pontocerebellar Hypoplasia type 1b (PCH1b), a disease characterized by atrophy of the pons and cerebellum. The missense mutations encode single amino acid changes in conserved regions of the EXOSC3 protein. How these amino acid substitutions confer tissue‐specific phenotypes is not known. One possible mechanism underlying the distinct disease phenotypes could be a decrease in the interaction of the RNA exosome complex with cofactors that confer specificity for RNA targets. However, most studies that identify and characterize RNA exosome cofactors have been carried out in budding yeast and thus, do not provide insight into whether tissue‐specific cofactors could exist. Our studies use immunoprecipitation from neuronal cell culture (N2A) and relevant mouse tissues to define RNA exosome cofactors. Biochemical experiments that employ cultured N2A cells were used to identify RNA exosome‐interacting proteins in both the nucleus and the cytoplasm. Preliminary results from this analysis reveal an association between the RNA exosome and a large complex of cytoplasmic tRNA ligase enzymes, which could link defects in tRNA maturation to disease pathology. We are extending these studies to explore the possibility of tissue‐specific cofactors by immunoprecipitating EXOSC3 and analyzing co‐purifying proteins from the mouse cerebellum (affected in disease) and cortex (unaffected). To complement these approaches to identify tissue‐specific RNA exosome cofactors, we are also exploring whether amino acid changes that are linked to disease alter cofactor interactions. These studies, which are being performed in N2A cells, have identified cofactors that show altered interactions with the EXOSC3 variants present in disease. These studies will provide insight into both the functional consequences of amino acid substitutions in the RNA exosome that cause disease and the role of cofactors in conferring RNA target specificity.

Identification of Tissue‐Specific RNA Exosome Cofactors as an Approach to Define Disease Mechanism

The RNA exosome, a 10‐subunit complex that mediates both RNA processing and degradation, plays a critical role in defining cellular expression profiles. This complex is ubiquitously expressed, essential, and critical for fundamental cellular functions, such as ribosomal RNA processing. Recent studies have linked mutations in genes encoding multiple subunits of the complex to tissue‐specific human disease. For example, mutations in the human EXOSC3 gene, coding for a subunit, cause Pontocerebellar Hypoplasia type 1b (PCH1b), a disease characterized by atrophy of the pons and cerebellum. The missense mutations encode single amino acid changes in conserved regions of the EXOSC3 protein. How these amino acid substitutions confer tissue‐specific phenotypes is not known. One possible mechanism underlying the distinct disease phenotypes could be a decrease in the interaction of the RNA exosome complex with cofactors that confer specificity for RNA targets. However, most studies that identify and characterize RNA exosome cofactors have been carried out in budding yeast and thus, do not provide insight into whether tissue‐specific cofactors could exist. Our studies use immunoprecipitation from neuronal cell culture (N2A) and mouse tissues to define RNA exosome cofactors. Biochemical experiments that employ cultured N2A cells identified RNA exosome interacting proteins that are enriched in both the nucleus and cytoplasm. Preliminary results from this analysis identify an association between the RNA exosome and a large complex of tRNA ligase enzymes, which could link defects in tRNA maturation to disease pathology. We are extending these studies to explore the possibility of tissue‐specific cofactors by immunoprecipitating EXOSC3 and analyzing co‐purifying proteins from the cerebellum (affected in disease) and the cortex (unaffected) in mouse brain. Finally, using CRISPR/Cas9 genome editing, we are testing whether the amino acid changes that occur in disease alter interactions with both known RNA exosome cofactors and interactors identified in our co‐immunoprecipitation studies. These studies will provide insight into both the functional consequences of amino acid substitutions in the RNA exosome that cause disease and the role of cofactors in conferring RNA target specificity.Support or Funding InformationR01GM130147This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Phenotype of c.92G >C (p.G31A) mutation in the EXOSC3 gene – 14 new cases and comparison with other causes of pontocerebellar hypoplasia type 1

PCH1 is a major cause of SMN-negative spinal muscular atrophy (SMA) and EXOSC3 mutations are the most common cause of PCH1. High prevalence of the p.G31A mutation of EXOSC3 gene was found recently, especially in the Roma ethnic minority.

Novel EXOSC3 mutation causes complicated hereditary spastic paraplegia.

We describe two pairs of siblings from a consanguineous family manifesting autosomal recessive hereditary spastic paraplegia caused by a novel mutation in the EXOSC3 gene, previously reported in pontocerebellar hypoplasia type 1. Clinical findings included delayed motor milestones, early-onset spastic paraplegia, variable cognitive disability, and cerebellar signs. Cerebral imaging demonstrated enlarged cisterna magna and mild hypoplasia and atrophy of the lower vermis with a normal pons. Genetic analysis using homozygosity mapping followed by whole exome sequencing identified homozygous c.571G>T; p.G191C mutation in the EXOSC3 gene. We suggest that EXOSC3 mutations may present not only as pontocerebellar hypoplasia type 1, but also as a complicated form of hereditary spastic paraplegia without pontine hypoplasia or atrophy.

EXOSC3 mutations in pontocerebellar hypoplasia type 1: novel mutations and genotype-phenotype correlations

BackgroundPontocerebellar hypoplasia (PCH) represents a group of neurodegenerative disorders with prenatal onset. Eight subtypes have been described thus far (PCH1-8) based on clinical and genetic features. Common characteristics include hypoplasia and atrophy of the cerebellum, variable pontine atrophy, and severe mental and motor impairments. PCH1 is distinctly characterized by the combination with degeneration of spinal motor neurons. Recently, mutations in the exosome component 3 gene (EXOSC3) have been identified in approximately half of the patients with PCH subtype 1.MethodsWe selected a cohort of 99 PCH patients (90 families) tested negative for mutations in the TSEN genes, RARS2, VRK1 and CASK. Patients in this cohort were referred with a tentative diagnose PCH type 1, 2, 4, 7 or unclassified PCH. Genetic analysis of the EXOSC3 gene was performed using Sanger sequencing. Clinical data, MR images and autopsy reports of patients positive for EXOSC3 mutations were analyzed.ResultsEXOSC3 mutations were found in twelve families with PCH subtype 1, and were not found in patients with other PCH subtypes. Identified mutations included a large deletion, nonsense and missense mutations. Examination of clinical data reveals a prolonged disease course in patients with a homozygous p.D132A mutation. MRI shows variable pontine hypoplasia in EXOSC3 mediated PCH, where the pons is largely preserved in patients with a homozygous p.D132A mutation, but attenuated in patients with other mutations. Additionally, bilateral cerebellar cysts were found in patients compound heterozygous for a p.D132A mutation and a nonsense allele.ConclusionsEXOSC3 mediated PCH shows clear genotype-phenotype correlations. A homozygous p.D132A mutation leads to PCH with possible survival into early puberty, and preservation of the pons. Compound heterozygosity for a p.D132A mutation and a nonsense or p.Y109N allele, a homozygous p.G31A mutation or a p.G135E mutation causes a more rapidly progressive course leading to death in infancy and attenuation of the ventral pons.Our findings imply a clear correlation between genetic mutation and clinical outcome in EXOSC3 mediated PCH, including variable involvement of the pons.

Exome sequencing in a family with intellectual disability, early onset spasticity, and cerebellar atrophy detects a novel mutation in EXOSC3

Whole exome sequencing in two-generational kindred from Bangladesh with early onset spasticity, mild intellectual disability, distal amyotrophy, and cerebellar atrophy transmitted as an autosomal recessive trait identified the following two missense mutations in the EXOSC3 gene: a novel p.V80F mutation and a known p.D132A change previously associated with mild variants of pontocerebellar hypoplasia type 1. This study confirms the involvement of RNA processing proteins in disorders with motor neuron and cerebellar degeneration overlapping with spinocerebellar ataxia 36 and rare forms of hereditary spastic paraplegia with cerebellar features.

Homozygous EXOSC3 Mutation c.92G→C, p.G31A is a Founder Mutation Causing Severe Pontocerebellar Hypoplasia Type 1 Among the Czech Roma

Pontocerebellar hypoplasia type 1 (PCH1) is characterized by cerebellar and anterior horn motor neuron degeneration and loss, signs of spinal muscular atrophy plus. Patients manifest severe perinatal weakness, hypotonia, and respiratory insufficiency, causing death frequently before the age of 1 year. Recently, causative mutations in EXOSC3 were reported in a majority of PCH1 patients, but the detailed clinical phenotype caused by EXOSC3 mutations, genotype-phenotype correlations, and prevalent mutations in specific ethnic groups is not yet known.Three unrelated Czech Roma patients with PCH1 were investigated clinically, electrophysiologically, neuroradiologically, and neuropathologically (patients 1 and 2). The entire coding region of the EXOSC3 gene, including the adjacent intron sequences, was sequenced in all three patients. The same mutation c.92G→C, p.G31A in EXOSC3 was found in all three affected patients in homozygous state and in heterozygous state in the parents from two of the families. Haplotype analysis with four flanking microsatellite markers showed identical haplotype in 9 out of 11 haplotypes carrying the c.92G→C, p.G31A mutation. Furthermore, four heterozygotes for this mutation were found in anonymous DNA samples from 90 unrelated Roma individuals. All four of these samples shared the same haplotype. No heterozygous sample was found among 120 anonymous DNA samples from Czech non-Roma individuals with no familial relation. It may therefore be concluded that EXOSC3 c.92G→C, p.G31A mutation is a founder mutation with high prevalence among the Czech Roma causing a similar and particularly severe phenotype of PCH1. These observations from the Czech Roma may have consequences also for other Roma from other countries.PCH1 caused by EXOSC3 founder mutation c.92G→C, p.G31A extends the list of autosomal recessive disorders rare among the general population but more frequent among Roma at least in the Czech Republic.

EXOSC3 mutations in isolated cerebellar hypoplasia and spinal anterior horn involvement

Pontocerebellar hypoplasia (PCH) type 1 is characterized by the co-occurrence of spinal anterior horn involvement and hypoplasia of the cerebellum and pons. EXOSC3 has been recently defined as a major cause of PCH type 1. Three different phenotypes showing variable severity have been reported. We identified a homozygous mutation [c.395A > C/p.D132A] in EXOSC3 in four patients with muscle hypotonia, developmental delay, spinal anterior horn involvement, and prolonged survival, consistent with the "mild PCH1 phenotype". Interestingly, isolated cerebellar hypoplasia limited to the hemispheres or involving both hemispheres and vermis was the main neuroradiologic finding, whereas the pontine volume was in the normal range for age. These findings strongly suggest that analysis of the EXOSC3 gene should be recommended also in patients with spinal anterior horn involvement and isolated cerebellar hypoplasia.

Pontocerebellar hypoplasia type 1

Pontocerebellar hypoplasia with spinal muscular atrophy, also known as PCH1, is a group of autosomal recessive disorders characterized by generalized muscle weakness and global developmental delay commonly resulting in early death. Gene defects had been discovered only in single patients until the recent identification of EXOSC3 mutations in several families with relatively mild course of PCH1. We aim to genetically stratify subjects in a large and well-defined cohort to define the clinical spectrum and genotype-phenotype correlation. We documented clinical, neuroimaging, and morphologic data of 37 subjects from 27 families with PCH1. EXOSC3 gene sequencing was performed in 27 unrelated index patients of mixed ethnicity. Biallelic mutations in EXOSC3 were detected in 10 of 27 families (37%). The most common mutation among all ethnic groups was c.395A>C, p.D132A, responsible for 11 (55%) of the 20 mutated alleles and ancestral in origin. The mutation-positive subjects typically presented with normal pregnancy, normal birth measurements, and relative preservation of brainstem and cortical structures. Psychomotor retardation was profound in all patients but lifespan was variable, with 3 subjects surviving beyond the late teens. Abnormal oculomotor function was commonly observed in patients surviving beyond the first year. Major clinical features previously reported in PCH1, including intrauterine abnormalities, postnatal hypoventilation and feeding difficulties, joint contractures, and neonatal death, were rarely observed in mutation-positive infants but were typical among the mutation-negative subjects. EXOSC3 mutations account for 30%-40% of patients with PCH1 with variability in survival and clinical severity that is correlated with the genotype.