Figure S1: Detailed analysis of synaptic defects in THOC mutant animals (Related to Figure 1)
(A) Quantification of synaptic density relative to wt for different THOC mutants and their corresponding cell-specific rescue.
(B) Quantification of synaptic size for different THOC mutants and their corresponding cell-specific rescue.
(C) Quantification of synapse intensity for different THOC mutants and their corresponding cell-specific rescue.
(A-C) N=10-20 animals at L4 stage; ****, p<0.0001, ***, p<0.001**, p<0.01, *, p<0.05, one-way Anova with Tukey post-hoc test.
(D) Quantification of synaptic density for L3 and L4 animals in wt and thoc-5 mutant background.
(E) Quantification of synaptic size for L3 and L4 animals in wt and thoc-5 mutant background.
(F) Quantification of synapse intensity for L3 and L4 animals in wt and thoc-5 mutant background.
(D-F) N=10-20 animals; ***, p<0.001, student t-test.
Averages and SEM are plotted in all graphes.
(G) thoc-1 mRNA quantification in wt and thoc-1 strain backgrounds show a 70% reduction in thoc-1 mRNA level in the mutant.
Figure S2: thoc mutation does not affect PDE neuronal health (Related to Figure 1)
(A) - (C) Visualization and quantification of mitochondria in PDE cell body (A) and axons (B), (C) in wt and thoc-5 mutants.
(D)-(I) thoc-5 mutation does not affect the localization, number and morphology of different organelle markers. (D) ribosomes, (E) ER, (F) Endosome, (G), (H) Golgi and (I) cilia.
Figure S3: THOC mutants are impaired in DA signaling and DA-dependent behavior (Related to Figure 1 & 2)
(A) and (B) Quantitative analysis of basal slowing response in the absence (A) or presence (B) of DA. Locomotion rates in the absence (black bars) or presence (white bars) of bacteria were calculated in a 20 second time window. Wt and cat-2 animals were included as controls. The percentage of slowing is shown on the right. N=30-60 animals; ***, p<0.001, **, p<0.01, one-way Anova with Tukey post-hoc test.
(C) Ca2+ response of PDE neuron during basal slowing response. Calcium trace and quantification of it is shown for wt and thoc-5 mutant animals.
Averages and SEM are plotted in all graphs.
Figure S4: Presynaptic transcript retention in the nucleus is rescued in thoc-5 creb mutant animals. (Related to Figure 3)
(A) Representative images for smFISH for presynaptic and control transcripts in wt (upper row) and thoc-5 (lower row) mutant animals. GFP: PDE cellbody; red: signal for FISH probes; blue: DAPI staining of nuclei. Scale bar represents 2μm.
(B) Total number of smFISH puncta in PDE is not changed by thoc-5 mutation.
(C) Gene enrichment analysis for down-regulated genes in thoc-5 mutant neurons.
(D) qPCR quantification of presynaptic as well control transcripts in wt and thoc-5 mutant strain backgrounds. N = 3 biological replicates for both genotypes. ****, p<0.0001, ***, p<0.001, **, p<0.01; one-way Anova with Tukey post-hoc test. Averages and standard deviations are plotted.
Figure S5: Putative disease-associated thoc-1 R123H mutation renders THOC-1 dysfunctional (Related to Figure 7)
(A) Sequence alignment of human, mouse and C. elegans THOC1. Human, ALS-associated mutations are highlighted. Red: conserved R123H mutation, Blue: other disease associated mutations on un-conserved residues.
(B) Integrated synaptic intensity is quantified for wt, thoc-1, thoc-1 Pdat-1::thoc-1R123H and thoc-1 Pdat-1::thoc-1V338I strain backgrounds. N=20 animals; ****, p<0.0001, one-way ANOVA with Tukey post-hoc test.
Averages and SEM are plotted.
Figure S6: CREB, SRF/UNC-120 and PAB-2 function with THOC to facilitate export of synaptic transcripts (Related to Figure 6)
(A) Integrated synaptic intensity is quantified for two lines, in which crh-1 is cell-specifically knocked-down in DA neuron by CRE-loxP strategy. N=10-20 animals;
(B) Quantification of nuclear transcripts in wt, thoc-5, thoc-5 crh-1 and crh-1 PDE neurons. N=10-50 animals.
(C) Integrated synaptic intensity is quantified for wt, thoc-5, thoc-5 unc-120 and unc-120 mutant animals. N=20-50 animals
(D) Integrated synaptic intensity is quantified for wt, thoc-5, thoc-5 pab-2 and pab-2 mutant animals. N=20-50 animals
(D) Quantification of nuclear transcripts in wt, thoc-5, thoc-5 pab-2 and pab-2 PDE neurons. N=10-50 animals. ***, p<0.001; one-way Anova with Tukey post-hoc test. Averages and SEM are plotted in all graphs.
****, p<0.0001, ***, p<0.001, **, p<0.01, *, p<0.05 one-way Anova with Tukey post-hoc test.
Figure S7: THOC-1 specifically interacts with CREB but not GFP and binds to presynaptic transcripts (Related to Figure 7)
(A) Localization of THOC and CRH-1a does not depend on each other. THOC component are not mis-localized in crh-1 mutant background. CRH-1A localization is not altered by thoc-5 mutation.
(B) Quantification of the interaction between THOC-1 and increasing GFP concentration. No significant interaction between those two proteins was detected even at very high GFP concentration in PFA fixed samples. These result further corroborate the specificity of the direct interaction between CREB and THOC-1.
(C) Relative IP efficiency of synaptic transcripts (orange bars) as well as other pan-neuronal and control genes (black). THOC-1 bound transcripts were pulled-down under wt conditions. Transcripts were then quantified by qPCR and normalized onto total mRNA.
(D) Correlation analysis between total mRNA level and IPed mRNAs for wt and crh-1 mutant animals. No correlation was detected for synaptic transcripts (orange bars) and control transcripts (black bars).
