Exosomal protein deficiencies : how abnormal RNA metabolism results in childhood-onset neurological diseases

Abstract

RNA metabolism is of critical importance for normal cellular functions and needs to be finely tuned in order to maintain stable conditions within the cell. The exosome complex is the most important RNA processing machinery, responsible for the correct processing of many different types of RNAs and interacting with different co-factors which bind and carry specific subtypes of RNA for degradation to the complex. Mutations in exosome complex subunits (EXOSC3, EXOSC8) were reported to cause severe childhood onset complex neurological disorders presenting with pontocerebellar hypoplasia type 1 (PCH1), spinal muscular atrophy (SMA) and central nervous system hypomyelination. We have recently identified a homozygous pathogenic mutation in RNA Binding Motif Protein 7 RBM7, a subunit of the nuclear exosome targeting (NEXT) complex in a single patient with SMA-like phenotype and proved that RBM7 is a novel human disease gene related to the exosome complex. In order to understand the disease mechanism in RBM7 deficiency and to explore the role of exosome complex in neurodevelopment, we performed gene expression studies (RT-PCR, RNA sequencing) in human cells of patients carrying mutations in EXOSC8 and RBM7. Furthermore we performed functional studies in zebrafish (D. rerio) by morpholino oligonucleotide mediated knock-down of rbm7, exosc8 and exosc3 and also by introducing pathogenic mutations in exosomal protein genes in zebrafish embryos by the CRISPR/Cas9 system. We showed that mutations in RBM7 and EXOSC8 mutant fibroblasts cause differential expression of several different transcripts, 62 of them being shared between the two cell lines. Altered gene expression of some AU-rich element containing genes may potentially contribute to the clinical presentation. Knock-down of rbm7, exosc8 and exosc3 caused impaired neurodevelopment in zebrafish, illustrated by abnormal growth of motor neuron axons and failure to differentiate cerebellar Purkinje cells. RT-PCR analysis in zebrafish showed a dramatic increase in expression of atxn1b (an AU-rich element containing homolog of the human ATXN1 gene) in rbm7, exosc8 and exosc3 downregulated fish, which may be responsible for the cerebellar defects. We have successfully introduced several germline mutations with CRISPR/Cas9 technology in rbm7. Phenotype of the F1 mutants is milder than what observed with the morpholino oligonucleotide injected fish. Mutants at a closer look do not show any morphological defect but further experiment may indicate similar characteristics to the morphants, although more iv subtle. Further studies on the CRISPR/Cas9 generated zebrafish models will extend our knowledge on the disease mechanisms caused by defective RNA metabolism.