RBM38 interacts with eIF4G and promotes target mRNA translation. (A) RBM38 can stimulate translation. 3′UTR tethering reporter assays (top, reporter design) provide evidence of the capacity of RBM38 to significantly enhance translation of an intronless reporter. (B) RBM38 overexpression promotes polyribosome formation, as evidenced by polysome profiling. (C-D) Specific and RNA-independent interaction between RBM38 and eIF4G. HA-tagged RBM38 was immunoprecipitated from MEL cells (C), and endogenous eIF4G was immunoprecipitated from primary fetal liver erythroblasts (D). The samples were resolved by SDS-PAGE followed by western blotting for the indicated proteins. (E) Validation of RBM38-mediated translational activation of target mRNAs. Selected mRNAs translationally activated or repressed with differentiation were tested for specific immunoprecipitation by an antibody against HA-RBM38. (F) RBM38 overexpression promotes translation of endogenous mRNA targets. The distribution of target or control mRNAs across a polysome gradient was determined in both the presence and the absence of RBM38 expression. (G) Global translation is compromised in RBM38-depleted erythroblasts. Erythroblasts transduced with different shRNA-expressing retroviruses were pulse-labeled with a methionine analog (HPG) and then subjected to a Click-iT assay to determine its rate of incorporation into polypeptides in TER119⁺ CD71⁺ erythroblasts (n = 3). mRNP, messenger ribonucleoprotein. *P < .05, Student t test.