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Fig. 1 | Acta Neuropathologica Communications

Fig. 1

From: RNA-recognition motif in Matrin-3 mediates neurodegeneration through interaction with hnRNPM

Fig. 1

Ubiquitous expression of MATR3 is toxic in Drosophila. a Schematic diagram of MATR3 protein domain architecture, consisting of two tandem RNA-recognition motifs (RRM1 and RRM2) and two Zinc Finger motifs (ZF1 and ZF2). ALS-associated mutations in MATR3 are spread across intrinsically disordered regions of the protein. b Immunoblot showing transgenic expression of UAS-FLAG-MATR3 wildtype and mutants – F115C and S85C. c Quantification of replicate western blots to confirm equal transgenic expression levels due to site-specific integration. α-tubulin is used as loading control (n = 3 per group; One-way ANOVA). d Constitutive ubiquitous expression of MATR3 wildtype and mutants, driven by Tub-Gal4, is toxic in Drosophila leading to complete lethality. Quantification of egg-to-adult viability showed that control (driver-alone) and transgenic UAS-EGFP expression had no effect on viability (n = 3, One-way ANOVA). e Schematic of conditional expression of MATR3 during development and in adults using the inducible driver, TubGS-Gal4, that is activated by RU486. f Representative immunofluorescence images of third-instar larval neuromuscular junction (NMJ) immunostained for presynaptic marker, HRP. Yellow arrows point to the synaptic boutons. g Quantification of boutons, normalized to surface area, showed reduced number of boutons in MATR3-expressing larvae (n = 6-8; One-way ANOVA). h Kaplan–Meier survival curve of adults ubiquitously expressing MATR3 showing statistically-significant reduction in longevity of MATR3-expressing flies compared to driver-alone control flies (n = 80; Log-Rank Mantel-Cox test) i Quantification of motor dysfunction in day-12 adults expressing MATR3, induced on day-1. (n = 25-40, One-way ANOVA). j Quantification of NP40-soluble and k NP40-insoluble MATR3 degradation in vivo by a pulse-chase assay at t = 0, 12, 24 and 48 h following inhibition of transgene production (n = 3-4 per group, One-way ANOVA). Statistical significance shown for F115C and S85C groups compared to WT at same time point. NP40-soluble and insoluble MATR3 mutants have a higher half-life compared to MATR3 WT. Error bars indicate S.E.M. *p < 0.05; **p < 0.01; ****p < 0.0001

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