Fig 2: Pharmaceutical exclusion of TyrRS from the nucleus partially rescues the toxicity of TyrRS mutants. a, b Subcellular localization of TyrRS after treatment with different concentrations of embelin (Emb) in HEK293T cells (14 mM DMSO) (a) and in flies pan-neuronally (nSyb-Gal4) expressing TyrRS (42 mM DMSO) (b). Emb was dissolved in DMSO, which has no effect on TyrRS nuclear localization. α-Tubulin and Lamin served as cytoplasmic and nuclear markers, respectively. n ≥ 3 independent biological replicates, one-way ANOVA with Dunnett Multiple Comparisons test. Bar charts presented as means ± s.e.m. c Pupae formation of Drosophila ubiquitously (Act5C-Gal4) expressing TyrRS-E196K after administration of different concentrations of embelin. Emb was dissolved in 0.3% DMSO (42 mM). Percentages indicate ratio of pupae expressing the TyrRS transgene or not. Dashed line, expected 50:50 pupation rate in the control condition (no transgene). Number of counted pupae (n) are indicated above the chart χ2-test. d Climbing performance of flies pan-neuronally (nSyb-Gal4) expressing TyrRS-E196K raised on food containing Emb, dissolved in 0.3% DMSO (42 mM). n ≥ 10 independent groups of flies. One-way ANOVA with Dunnett Multiple Comparisons test. Box plots show the median, 25–75% percentiles, and 1.5 interquartile range. Source data are provided as a Source Data file

Fig 3: Mutant and WT GARS and YARS proteins display similar subcellular localization in motor and sensory neurons.(a–h) GARS immunostaining reveals the subcellular localization of GARS proteins at third instar larval NMJs (a–d) and motor neuron cell bodies (e–h) upon expression in motor neurons (OK371-GAL4). (i–t) Subcellular localization of YARS proteins at third instar larval NMJs (i–l; nsyb-GAL4), motor neuron cell bodies (m–p; nsyb-GAL4) and class IV multidendritic sensory neurons (q–t; ppk-GAL4), as revealed by YARS immunostaining. Scale bars, 20 μm (a–p) and 100 μm (q–t).

Fig 4: Mutant TyrRS induces broad transcriptional changes in Drosophila brain tissues. a Volcano plot of log2 fold change versus mean comparing the TyrRS-WT (nSyb>TyrRS-WT) with the control condition (nSyb>+). b Enriched motifs for the dE2F1 and gem transcription factors identified in the upregulated and downregulated genes of the TyrRS-WT condition, respectively. E-score, enrichment score. c Volcano plot of log2 fold change versus mean comparing the TyrRS-E196K (nSyb>TyrRS-E196K) with the control condition. d Venn diagram displaying the overlap between TyrRS-WT and TyrRS-E196K differentially expressed genes. RF, representation factor. p, associated probability. e Heat map of top 151 differentially expressed genes in the TyrRS-E196K specific 415-gene cluster from brains of Drosophila pan-neuronally (nSyb-Gal4) expressing the indicated transgenes. High expression is shown in red and low expression is shown in blue (z-scores). f TFs identified in the TyrRS-E196K specific gene cluster. TFs associated with the downregulated genes are depicted in blue and those associated with the upregulated set are shown in red. The overlapping TFs are indicated in yellow

Fig 5: Modulation of E2F1 activity does not rescue disease hallmarks in the TyrRS Drosophila model. a–c Retinas of control flies (GMR-Gal4>+) (a), flies expressing TyrRS-WT (b), and TyrRS-E196K (c) do not display a rough eye phenotype. d–l Retinas of flies with a control or TyrRS background that overexpress dE2F1 (d–f), Dp (g–i), and the combination of both transcription factors (j–l). Scale bars, 50 µm. m Acetylation of E2F1 after activators of Sirtuin1 (resveratrol (Res)) and HDAC1 (dexamethasone (Dex)) were administered to HEK293T cells expressing different TyrRS alleles. n = 3 independent biological replicates, one-way ANOVA with Dunnett Multiple Comparisons test. Bar charts are presented as means ± s.e.m. n Pupae formation as an indicator of developmental lethality when Drosophila ubiquitously expressing (Act5C-Gal4) TyrRS-E196K were grown on resveratrol or dexamethasone. Res and Dex were dissolved in 0.3% DMSO (42 mM). Number of counted flies (n) are indicated above the chart, χ2-test. o Climbing performance of flies pan-neuronally expressing (nSyb-Gal4) TyrRS-E196K when grown on food containing a combination of resveratrol and dexamethasone. Res and Dex were dissolved in 0.3% DMSO (42 mM). n ≥ 10 independent groups of flies, one-way ANOVA with Dunnett Multiple Comparisons test. Box plots show the median, 25–75% percentiles, and 1.5 interquartile range. Source data are provided as a Source Data file