RESUMEN
Translation initiation is the rate-limiting step of protein synthesis and is the main target of translation regulation. RNA-binding proteins (RBPs) are key mediators of the spatiotemporal control of translation and are critical for cell proliferation, development, and differentiation. We have previously shown that HuD, one of the neuronal RBPs, enhances cap-dependent translation through the direct interaction with eukaryotic initiation factor 4A (eIF4A) and poly(A) tail using a HeLa-derived in vitro translation system. We have also found that translation stimulation of HuD is essential for HuD-induced neurite outgrowth in PC12 cells. However, it remains unclear how HuD is involved in the regulation of translation initiation. Here, we report that HuD binds to eukaryotic initiation factor 3 (eIF3) via the eIF3b subunit, which belongs to the functional core of mammalian eIF3. eIF3 plays an essential role in recruiting the 40S ribosomal subunit onto mRNA in translation initiation. We hypothesize that the interaction between HuD and eIF3 stabilizes the translation initiation complex and increases translation efficiency. We also showed that the linker region of HuD is required for the interaction with eIF3b. Moreover, we found that eIF3b-binding region of HuD is conserved in all Hu proteins (HuB, HuC, HuD, and HuR). These data might also help to explain how Hu proteins stimulate translation in a cap- and poly(A)-dependent way.
Asunto(s)
Factor 3 de Iniciación Eucariótica , Factores Eucarióticos de Iniciación , Animales , Humanos , Ratas , Factor 3 de Iniciación Eucariótica/genética , Factor 3 de Iniciación Eucariótica/metabolismo , Factores Eucarióticos de Iniciación/metabolismo , Neuronas/metabolismo , Factor 3 Procariótico de Iniciación/genética , Factor 3 Procariótico de Iniciación/metabolismo , Unión Proteica , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Células HeLaRESUMEN
The RNA-binding protein HuD/ELAVL4 is essential for neuronal development and synaptic plasticity by governing various post-transcriptional processes of target mRNAs, including stability, translation, and localization. We previously showed that the linker region and poly(A)-binding domain of HuD play a pivotal role in promoting translation and inducing neurite outgrowth. In addition, we found that HuD interacts exclusively with the active form of Akt1, through the linker region. Although this interaction is essential for neurite outgrowth, HuD is not a substrate for Akt1, raising questions about the dynamics between HuD-mediated translational stimulation and its association with active Akt1. Here, we demonstrate that active Akt1 interacts with the cap-binding complex via HuD. We identify key amino acids in linker region of HuD responsible for Akt1 interaction, leading to the generation of two point-mutated HuD variants: one that is incapable of binding to Akt1 and another that can interact with Akt1 regardless of its phosphorylation status. In vitro translation assays using these mutants reveal that HuD-mediated translation stimulation is independent of its binding to Akt1. In addition, it is evident that the interaction between HuD and active Akt1 is essential for HuD-induced neurite outgrowth, whereas a HuD mutant capable of binding to any form of Akt1 leads to aberrant neurite development. Collectively, our results revisit the understanding of the HuD-Akt1 interaction in translation and suggest that this interaction contributes to HuD-mediated neurite outgrowth via a unique molecular mechanism distinct from translation regulation.