Regulation of Ribosome Biogenesis in Hematopoietic Stem Cells

摘要 严格调控的蛋白质合成速率对于造血干细胞（HSC）的维持和功能至关重要。 核糖体蛋白或影响核糖体生物发生的基因突变引起“核糖体病”，这是一类 骨髓衰竭（BMF）综合征。如Shwachman-Diamond综合征（SDS）所突出说明的， BMF疾病伴进行性造血干细胞和祖细胞（HSPC）衰竭和易患 骨髓恶性肿瘤是由生殖系双等位基因突变驱动的组装因子所必需的， 60 S核糖体亚基的成熟。然而，在HSC中如何调控核糖体组装仍然很差 理解，因为是它的造血功能障碍的贡献。重要的是，除了异基因干细胞 在移植中，不存在减轻BMF中HSPC缺陷的治疗干预。本申请是 基于我们的新研究，发现了E3泛素连接酶HectD 1在调节HSC中的新作用， 通过核糖体生物合成发挥作用。Hectd 1缺陷型HSC在移植能力方面表现出显著缺陷， 自我更新，伴随着全球蛋白质合成的减少。HSC的机制 Hectd 1缺乏时的功能障碍与通过泛素化和 调节ZNF 622的稳定性，ZNF 622是60 S大核糖体成熟的关键生物发生因子， 细胞质中的亚基。HectD 1的缺失导致ZNF 622和抗结合因子的积累 eIF 6作用于60 S亚基，降低了80 S单体与60 S的比例，与亚基连接缺陷一致 与SDS样疾病有关。重要的是，在Hectd 1缺陷细胞中敲低ZNF 622， 合成和HSC重建能力。这一发现代表了一个罕见的体内遗传抑制的例子 与核糖体生物合成功能障碍相关的HSC缺陷。这一新途径的意义在于， HSC衰竭的病因学和“核糖体病”的临床治疗，要求详细的机制 认识在此，我们对HectD 1和ZNF 622在细胞凋亡中的作用进行了全面深入的分析。 核糖体生物发生和HSC。目的1：研究HectD 1和ZNF 622在HSC中的作用 以及它们如何相互作用以调节HSC功能，使用互补遗传学，基因组学和 生物化学方法。目的2：系统分析HectD 1/ZNF 622是否影响细胞的各个方面， 蛋白质翻译控制。此外，我们将进行定量蛋白质组学，以评估核糖体水平或 核糖体组成受Hectd 1/ZNF 622损失的影响。在目标3中，我们将探讨潜在的失调， HECTD 1和ZNF 622在人类BMF综合征中的作用，并探索靶向ZNF 622治疗BMF综合征的可能性。 用功能失调的核糖体生物合成治疗BMF。我们的研究揭示了一个以前未被重视的作用 泛素化通过控制核糖体生物合成因子来调节HSC功能， 在核糖体病中我们的研究结果可能会对调节免疫球蛋白的治疗潜力产生重大影响。 泛素化和/或核糖体生物发生因子在恢复BMF综合征中HSC功能中的作用。

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 ribosome biogenesis factors, which are dysregulated in ribosomopathies. Our findings will likely have significant impact on the therapeutic potential of modulating ubiquitination and/or ribosome biogenesis factors in restoring HSC functions in BMF syndromes.