Transcriptional profiling (RNA-Seq) and a forward genetic mutagenesis screen identify candidate genes required for TCS.

(A) RNA-Seq scatterplot showing log 2-fold change in expression levels of genes (P hsp-90int compared to hsp-90control. (B) RNA-Seq scatterplot showing log 2-fold change in expression levels of genes (P hsp-90neuro compared to hsp-90control. (C) GO enrichment analysis of genes up-regulated by TCS in hsp-90int. GO categories are reported as the -log 10 transformation of the P value. (D) GO enrichment analysis of genes up-regulated by TCS in hsp-90neuro. (E) Venn diagram analysis of genes up-regulated in both hsp-90int and hsp-90neu. P value overlap = 0.022 was calculated using probability mass function of overlap size based on hypergeometric distribution. (F) hsp-90int mutants isolated after an EMS mutagenesis screen showing reduced global hsp-70 mRNA expression that are comparable to hsp-90control control animals. WGS analysis identified 40 mutated genes in the isolated hsp-90int mutants. See also S2 Fig. Bar graphs represent the average of 3 biological replicates of >50 animals per strain; error bars represent SEM of the 3 biological replicates. Significance compared to the original strain hsp-90int (pre-mutagenesis) was determined using Student’s t test. ***P P G) Flowchart of identification of the 72 candidate genes resulting from the RNA-Seq analysis (17 genes), the mutagenesis and WGS analysis (40 genes), and proteostasis regulators and txt-1 interactors (15 genes) that were taken further for a tissue-specific RNAi screen. Note that only 13 (out of 17) candidates of the RNA-Seq analysis (S1 Table) and 31 candidates (out of 40) of the WGS analysis (S2 Table) could be used for further RNAi analysis, as some candidate genes were not present in the genome-wide Ahringer RNAi library. Source data for Fig 3A–D and 3F is provided in S3 Data. GO, Gene Ontology; RNAi, RNA interference; TCS, transcellular chaperone signaling; WGS, whole-genome sequencing.