SCIENTIFIC ABSTRACT Erythropoiesis is guided by a complex interplay of signaling cues triggering expression of gene networks necessary for production of mature red blood cells. Rapid response to these signals in erythroid precursors temporally orchestrate functions required for erythroid differentiation. Timely and appropriate selection of mRNAs for translation by the ribosome is essential for maintaining the balance between maintaining erythroid precursors differentiation. This concept, known as translational control governs efficiency of mRNA translation and thus plays a crucial role in regulating responses to extracellular cues such as anemia and hypoxia. Failure of the ribosomal machinery to regulate this process underlies the ribosomopathies, a set of human diseases resulting from ribosomal mutations leading to profound consequences for hematopoietic maturation. This proposal focuses on outstanding, fundamental questions in how translational control 1.) directs mRNA selection in normal erythropoiesis and 2.) is impaired in ribosomopathies in response to signaling cues.
Aim 1 will explore how eIF4E, a key factor in translation initiation is repressed in a dynamic, cell-intrinsic manner to direct translation of a specific set of transcripts. Failure to repress eIF4E activity in maturing erythroblasts leads to inappropriate expression of genes necessary for maintenance of early erythroid precursors and impaired differentiation.
This aim will mechanistically decipher motifs in target mRNA and decipher how eIF4E specifically recognizes these transcripts for ribosomal recruitment. Dependence of tight regulation of eIF4E on erythropoiesis will be tested in vivo employing a novel mouse model utilizing doxycycline-responsive expression of eIF4E localized to specific phases of hematopoietic maturation. A paradigm example of ribosomopathies, Diamond Blackfan Anemia (DBA) results in erythroid aplasia and failure to respond to erythropoietin (Epo), a key signal in differentiation of early erythroid precursors. However, why hematopoietic precursors in DBA fail to generate the appropriate nascent gene expression response to these cues is still poorly understood.
Aim 2 will use employ a universally-applicable, mass spectrometry-based method (OPP-ID) pioneered by Dr. Forester to characterize the nascent proteomic landscape in healthy and DBA-derived iPSCs in response to erythropoietin. Identification of impaired proteomic responses in DBA iPSCs will give insight into how specific ribosomal mutations prevent translation of key genes crucial in early differentiation. This technology will be applied to understand how dexamethasone, a known mediator of hematopoietic rescue of unclear mechanism, remodels the erythroid proteome in response to Epo. Successful completion will result in both a unique contribution into the fields of translational control and erythropoiesis as well as a novel approach to understanding how the nascent translatome is impaired in ribosomopathies. Accomplishment of this proposal is crucial in Dr. Forester?s long-term goals of becoming an independently funded researcher and an expert in the field of proteomic networks governing early hematopoiesis.
Erythropoiesis is a fundamental biological process which is constantly responding to signals instructing new genes to be translated into proteins crucial for production of mature red blood cells. A powerful, yet poorly understood mechanism cells use to control gene expression exists at the protein synthesis machinery known as the ribosome. This research aims to understand how genes required for erythropoiesis are directed to the ribosome for production of specific proteins and how this process is deregulated in patients with ribosomal mutations lead to profound anemia.