Fig 2: Simultaneous knock down of exosc8/mbpa/p53 in zebrafish.(a) Representative graph shows the survival rate of un-injected, mpba+p53, exosc8+p53 and combined mbpa+exosc8+p53 MO injections of the Tg(islet-1:GFP) strain at 48 hpf (summary of 3 experiments). The survival rate increased after triple MO injections compared with exosc8+p53 knockdown embryos. (b) Top row: morphology of the severe embryos at 48 hpf injected with exosc8+p53 and exosc8+mbpa+p53. Bottom row: brain abnormalities of exosc8+p53 and exosc8+mbpa+p53 morphants (Tg(islet-1:GFP) strain) at 48 hpf. Dorsal views of GFP positive neurons in the midbrain and the hindbrain indicate abnormal brain structures in severe exosc8+p53 morphants. Improved cranial neuron structure was detected in severe mbpa+exosc8+p53 MO injected embryos. The same result has been reproduced in three separate experiments with equal MO doses, on the same clutch of embryos.

Fig 3: MBP and acetylated tubulin staining after knock down of the zebrafish ortholog: exosc8.(a) exosc8 MO injected larvae were analyzed for expression of AU-rich mRNAs during 48 hpf by real-time PCR. At 16 hpf expression of mbp1 and plp1 was increased in embryos with an abnormal phenotype, with a similar increase in mbp2 observed at 24 hpf. Despite this initial increase, by 48 hpf there is a dramatic decrease in mbp1 and plp1 expression in larvae with a moderate and severe phenotype. Each bar or severity group at different timepoints represents a number of 15–20 embryos. Statistically significant changes (P<0.05) are marked with *. Unpaired t-test was used for statistical analysis. Error bars represent s.d. of three experimental repeats. (b) Un-injected control larvae and exosc8 MO injected larvae were analyzed for myelination at 96 hpf. Larvae were stained with antibodies against the zebrafish MBP and against acetylated tubulin. Left column: overlay, MBP staining in red, acetylated tubulin staining in green; middle column: MBP staining; right column: acetylated tubulin. Top row: tail of control larva: motor axons in each somite are clearly visible and myelinated at 96 hpf. Second row: tail of MO injected larva with a moderate phenotype: the spinal cord is curved and has an irregular structure. Motor axons in the somites are either very short (arrowhead) and thin or missing completely and are not MBP-positive. Third row: un-injected control larva, posterior lateral line, intact myelin. Bottom row: exosc8 MO injected larva with moderate phenotype: the lateral line is present (green acetylated tubulin signal) but the myelination of its neurons is interrupted (arrowhead). Scale bar, 100 µm. (c) Myelin staining of the lateral line was studied in control un-injected and exosc8 MO injected zebrafish larvae at 4.5 dpf. Representative images of the analyzed embryos are shown on the top (scale bar, 0.25 mm) and transverse sections of the embryos are shown on the bottom. In the control larvae the myelinated lateral line is present at both sides (white arrowheads). However, no myelination of the lateral line was detected in the exosc8 MO injected larvae (white arrowheads). Higher magnifications are shown in the upper right hand corners. Scale bar, 100 µm. (d) Representative EM pictures of the myelin sheath at the lateral line in un-injected and exosc8 MO injected zebrafish larvae at 4 dpf. Black arrows indicate the myelin sheet around the axon. Scale bar, 500 µm.

Fig 4: Validation of EXOSC8rs117135638 as a GEMINI vulnerability.a Schematic of EXOSC8 SNP rs117135638 locus showing target sites for positive control, non-allele specific (NA) sgRNA and experimental, allele-specific (AS) sgRNA. Alleles appear in bold. b Crystal structure of EXOSC8 gene product, Rrp4389 (gray) shows the amino acid encoded by rs117135638 (teal) lies on the surface of the Rrp43 protein near the interface with exosome complex subunit Mtr3 (orange). c Disruption of EXOSC8 in patient-derived EXOSC8 resistant (EXOSC8R) or EXOSC8 sensitive (EXOSC8S) cells expressing EXOSC8 non-allele specific (NA) positive control sgRNA or allele-specific (AS) experimental sgRNA. Unaltered alleles (black), alleles with in-frame insertions or deletions (gray), and alleles with frameshift alterations (yellow) were assessed by deep sequencing of EXOSC8 four days post-infection with sgRNA. d Immunoblot of EXOSC8 protein levels in indicated patient-derived and isogenic cell lines expressing LacZ, EXOSC8 NA, or EXOSC8 AS sgRNA (n = 2 technical replicates of 1 biological sample). e Representative growth curves of indicated patient-derived and isogenic cell lines expressing LacZ (black), EXOSC8 NA (red), or EXOSC8 AS (blue) sgRNA, as measured by CellTiter-Glo luminescence, relative to day of assay plating. n = 5 technical replicates. Data are presented as mean values ± s.d. See Supplementary Fig. 4 for additional biological replicates. f Immunoblot of EXOSC8 protein levels in indicated isogenic cell lines expressing LacZ, EXOSC8 NA, or EXOSC8 AS sgRNA (n = 2 technical replicates of 1 biological sample). g Representative growth curves of indicated isogenic cell lines expressing LacZ (black), EXOSC8 NA (red), or EXOSC8 AS (blue) sgRNA, as measured by CellTiter-Glo luminescence, relative to day of assay plating. n = 5 technical replicates. Data are presented as mean values ± s.d. See Supplementary Fig. 4 for additional biological replicates. Source data for Fig. 3c–g are provided as a Source Data file.

Fig 5: RT-PCR studies in patient myoblasts, control myoblasts and human oligodendroglia cells and immunohistochemistry and immunoblotting for myelin basic protein (MBP) in human oligodendroglia cells.(a) Quantitative PCR of MBP, PMP22 and EIF2B2 in patient and control myoblasts. The gene expression of MBP shows significant increase (fold change>2.99, P=0.0055) while no other gene expression is altered. C. M., control myoblasts; P. M., P1-V:10 myoblasts. (b) Relative quantification of MBP expression in human myoblasts treated with EXOSC8 siRNA. Downregulation of EXOSC8 resulted in increased expression of MBP and MOBP, but not in the non-AU-rich control genes PMP22 and EIF2B2. C. M.,control myoblasts; CNTR., control; PP. M., P1-V:10 myoblasts;. (c) RT-PCR detected >100-fold increase of MBP mRNA in human oligodendroglia cells after EXOSC8 downregulation, but no significant change was observed in mRNAs of the non AU-rich control genes PMP22 and EIF2B2. Unpaired t-test was used for statistical analysis. Statistically significant changes are marked with *. Error bars show standard deviation of three experimental repetitions. (d) Quantitative PCR of SMN1, BICD2 and IGHMBP2 in patient and control fibroblasts. The gene expression of SMN1 shows significant increase (~fourfold change, P=0.01984) while no other gene expression is altered. C. F., control fibroblasts; P. F., P3-II:1 fibroblasts. (e) Quantification of ARE-containing and non-ARE-containing genes in human fibroblasts after 3 days EXOSC8 downregulation detected significant increase in SMN1 and MBP. (f) Immunoblotting confirmed that EXOSC8 siRNA downregulation resulted in severe depletion of this protein in both myoblasts (M) and (g) fibroblasts (F). (h) Immunoblotting (repeated three times) confirmed that siRNA downregulation of EXOSC8 resulted in increased MBP protein levels in human oligodendroglia cells. CNTR., control; NT siRNA, non-targeting siRNA. (i) Quantification of MBP protein expression in differentiated oligodendroglia cells after EXOSC8 downregulation. (j) Double immunolabelling of differentiated human oligodendroglia cells for MBP (green channel) and EXOSC8 (red channel). Left column: non-targeting control transfected cells. NT siRNA, non-targeting siRNA. EXOSC8 localized to the nucleus and MBP is distributed throughout the cytosol in EXOSC8 ablated cells. Right column: EXOSC8 staining confirms successful siRNA-mediated downregulation and increased MBP expression. Scale bar, 20 µm.