1. The DHX15–CMTR1 interaction inhibits translation of a subset of genes.
The DHX15–CMTR1 interaction inhibits translation of a subset of genes. (A) Extracts from HCC1806 cells transfected with pCDNA5 HA-CMTR1, 2L/A, or vector control (V) were centrifuged through 10%–50% sucrose gradients. 259 nm absorbance was determined across the gradient in four independent experiments. Representative experiment presented. The fraction of the gradients analysed by RNAseq is indicated by shaded boxes. The identities of the peaks in the polysome profiles is indicated. (B) Cell extracts were analysed by Western blot. (C) Average and standard deviation of ratio of polysome to mononosome absorbance for four independent experiments. RNAseq analysis was used to determine uniquely aligned reads per gene (transcript isoforms collapsed) in RNA and polysome samples from HCC1806 cells expressing HA-CMTR1 WT and 2L/A (n = 2). (D) Scatter plot of log2 transformed total counts per million (log2CPM) in total RNA. (E) Scatter plot of log2-fold change (FC) in 2L/A/WT for total RNA and polysomal RNA. Significantly regulated genes determined by a negative binomial rest in EdgeR in red. (F) Scatter plot of log2-transformed counts per million (log2CPM) in polysomal RNA. (G) Polysomal mRNA levels quantified by RT–PCR. Average and standard deviation for three independent experiments. t test was performed ***P-value < (H) Western blot analysis of proteins in HCC1806 cells expressing pCDNA5 HA-CMTR1, 2L/A, or vector control (V). Charts represented average protein signal in Western blots and standard deviation for three independent experiments. Quantitation performed in ImageJ software.
Francisco Inesta-Vaquera et al. LSA 2018;1:e
© 2018 Inesta-Vaquera et al.