Regulation of eIF4E Guides a Unique Translational Program to Steer Erythroid Maturation

Erythropoiesis is an intricately orchestrated process responsible for rapidly responding to an array of signaling cues to direct cell fates. While gene expression during erythropoiesis has mainly been studied at the transcription level, regulation of gene expression at the level of translation is still poorly understood. As translational control is one of the fastest steps to regulate protein abundance in the cell, we hypothesize that this mechanism plays an important role to rapidly control protein levels in response to extracellular cues during erythroid differentiation. However, outstanding questions remain on how translational control regulates specific gene programs in early erythroid states including 1.) how mRNA are selected for translation in normal erythropoiesis and 2.) how components of the translational machinery are regulated by upstream signaling pathways to orchestrate the translational landscape. In addressing these questions, we have nuncovered a dynamic interplay between Eukaryotic Initiation Factor 4E (eIF4E), the major mRNA cap binding protein that controls translation initiation and its repressor protein, 4EBP1 in erythropoiesis. Specifically, using in vivo phospho-flow cytometry analysis of the eIF4E-4EBP1 axis, eIF4E activity is high in early erythroid phases and is repressed by 4EBP1 in order to allow erythroid maturation. Surprisingly, high eIF4E activity in early erythroid precursors occurs without an increase in global protein synthesis. Utilizing a model of CD34+ human cord blood cells (HUDEP-2), we show that constitutive overexpression of eIF4E impaired erythropoietic maturation. To capture the specific proteins potentially regulated by eIF4E activity during erythropoiesis, we performed quantitative TMT mass spectrometry during HUDEP-2 erythroid differentiation. Our results revealed that eIF4E controls a specific key network of genes necessary for maintenance of early erythroid precursors. By analyzing the 5' untranslated region (5'UTR) of the eIF4E-dependent mRNA network, we identify a highly conserved, CT-rich motif which is required for these mRNAs to be more efficiently translated with increasing eIF4E levels. These results demonstrate wide-spread translational control of CT-rich mRNAs by eIF4E during early erythropoiesis. We are currently employing DMS-MaPseq to understand whether these motifs are also part of structured RNA elements that confer sensitivity to eIF4E levels. We have further extended these findings to a novel transgenic mouse model we have developed that allows in vivo assessment of increased eIF4E temporally at concise phases of erythroid maturation. Understanding this balance of eIF4E activity provides a novel insight into how of translational control dictates gene expression to determine phases of maturation in a crucial differentiation process.