DHX34 regulates NMD and pre-mRNA splicing. (A) Outline of the experimental design for the analysis of changes in gene expression and alternative splicing upon DHX34 knockdown. RNA-seq was performed from HeLa cells depleted of DHX34 (siDHX34) or transfected with nontargeting siRNA pools (ctrl). Sequencing reads were mapped using Salmon and differential gene expression (DGE) was performed with Sleuth. Splicing changes were detected with SUPPA2. (B) Volcano plot of DGE changes upon DHX34 depletion are indicated by altered b-value and –log10 adjusted P-value. (C) Splicing changes upon DHX34 depletion. Significant splice changes detected with SUPPA2 algorithm are depicted in red (dPSI > 0.05, P ≤ 0.05). Bar plot indicates pre-mRNAs that show AS changes as well as changes in gene expression: up-regulated expression (DGE UP, dark red), down-regulated (DGE DOWN, blue), not changed (gray). (D) Pie chart showing different types of alternative splicing events detected with SUPPA2. (E) Validation of cassette exon splice changes for ACOT9, CLSTN1, MAP2K7, MYL6, and MYOF transcripts by RT-PCR in HeLa cells depleted for DHX34 or treated with nontargeting siRNA (control). Dark gray arrow indicates transcript variant with included exon, light gray with excluded exon. Means from four individual data points obtained by RT-PCR using Bioanalyzer are plotted with standard deviations as error bars.
