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. 2020 Aug 18;8:138. doi: 10.1186/s40478-020-01021-5

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© The Author(s) 2020

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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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