Abstract Tightly-regulated protein synthesis rates are critical for hematopoietic stem cell (HSC) maintenance and function. Mutations in ribosome proteins or genes that affect ribosome biogenesis cause “ribosomopathies”, a class of bone marrow failure (BMF) syndromes. As prominently illustrated by Shwachman-Diamond Syndrome (SDS), a BMF disease with progressive hematopoietic stem and progenitor cell (HSPC) failure and predisposition to myeloid malignancies, is driven by germline biallelic mutations in the assembly factors essential for the maturation of the 60S ribosome subunit. However, how ribosome assembly is regulated in HSCs remains poorly understood, as is its contribution to hematopoietic dysfunction. Importantly, other than allogeneic stem cell transplantation, therapeutic interventions that mitigate the HSPC defects in BMF do not exist. This application is based on our new studies that uncovered a novel role for the E3 ubiquitin ligase, HectD1, in regulating HSC function via ribosome biogenesis. Hectd1-deficient HSCs exhibit a striking defect in transplantation ability and self-renewal, concomitant with a reduction in global protein synthesis. The mechanism underlying HSC dysfunction upon Hectd1 deficiency is directly linked to aberrant ribosome assembly by ubiquitinating and regulating the stability of ZNF622, a critical biogenesis factor for the maturation of the 60S large ribosomal subunit in the cytoplasm. Depletion of HectD1 led to an accumulation of ZNF622 and the anti-association factor eIF6 on the 60S subunit, decreased 80S monosome to 60S ratio, consistent with a subunit joining defect associated with SDS-like diseases. Importantly, knockdown of ZNF622 in Hectd1-deficient cells restored protein synthesis and HSC reconstitution capacity. This finding represents a rare in vivo example of genetic suppression of HSC defects associated with dysfunctional ribosome biogenesis. The implications of this novel pathway to the etiology of HSC failure and clinical treatment of “ribosomopathies”, mandates detailed mechanistic understanding. Here, we propose comprehensive and in-depth analyses on the role of HectD1 and ZNF622 in ribosome biogenesis and HSCs. In aim 1, we propose to investigate the roles of HectD1 and ZNF622 in HSCs and how they interact to regulate HSC function, using a combination of complementary genetics, genomics, and biochemical approaches. In aim 2, we will systematically analyze if HectD1/ZNF622 affects different aspects of protein translation controls. Moreover, we will perform quantitative proteomics to assess if ribosome levels or ribosome composition is affected by Hectd1/ZNF622 loss. In aim 3, we will interrogate potential dysregulation of HECTD1 and ZNF622 in human BMF syndromes and explore therapeutic potential of targeting ZNF622 for the treatment of BMF with dysfunctional ribosome biogenesis. Our study implicates a previously unappreciated role of ubiquitination in regulating HSC function via controlling riboso...