Fig. 4: 4EHP binds to NELF-E mRNA, dependent on its cap-binding domain, to regulate NELF-E expression.

a The design of the TRIBE experiment to identify 4EHP-binding mRNAs. The RNA editing enzyme, ADAR, was fused to either the wild-type 4EHP or to the cap binding-deficient 4EHP ^W114A mutant. These chimera-proteins are designed to edit the mRNAs (from A to I) that they bind. b A plot of mRNAs preferentially edited by 4EHP-ADAR. The binding score on the y axis shows the log-likelihood of mRNA editing by 4EHP wild type-ADAR on a specific nucleotide divided by that from the equivalent 4EHP ^W114A mutant. Highlighted are two different edited sites on NELF-E and NELF-A. c The enriched GO Terms of the 4EHP targets with binding scores above 10. d A schematic diagram of the NELF-E mRNA and the relative positions of the sites edited by 4EHP wild type-ADAR. Gray shows the UTRs, and black indicates the coding sequence. e NELF-E transcript levels in control and 4EHP RNAi RNA-seq dataset with three biological replicates per genotype (n = 3). A two-sided t test was used to determine significance. Data presented are mean values +/− SE. f Anti-NELF-E (top gel) and anti-tubulin (bottom gel) western blots from fat bodies of control (w¹¹¹⁸), or with 4EHP (VDRC #38399) or NELF-E RNAi (VDRC #21009). g Quantification of relative NELF-E protein band intensities as compared to that of the control sample across three biological replicates (each averaged from technical duplicate, n = 3). Data presented are mean values +/− SD. An ordinary one-way ANOVA followed by Tukey’s multiple comparisons test was used to assess statistical significance. p-values are indicated. ns = not significant.