Hematopoietic Stem Cells Supporting Fetal Erythropoiesis Are Differentially Regulated By Small and Large Ribosomal Subunits

Diamond Blackfan anemia (DBA) is a congenital bone marrow failure syndrome commonly associated with mutations or deletions of ribosomal genes, leading to protein haploinsufficiency and resulting in nucleolar stress and p53 activation. Clinically, DBA usually manifests after birth and is characterized by macrocytosis, reticulocytopenia and a paucity of erythroid precursors in the bone marrow. Patients can also present with additional defects in other hematopoietic lineages, suggesting a defect in hematopoietic stem and/or progenitor cells. Notably though, it is still unclear whether these defects are common to all ribosomal protein (RP) mutations or specific to large vs.small ribosomal subunits. Furthermore, the role of RPs in fetal hematopoiesis is poorly understood due to the lack of clinically relevant mouse models for ribosomopathies. To address these questions, we generated Rps19 fl/fl and Rpl5fl/fl conditional mouse models - representing the most mutated genotypes in DBA - using CRISPR/Cas9 technology and crossed them to a Vav-iCre mouse. Vav-iCre;Rps19fl/+ mice recapitulated clinical features of DBA, including macrocytic anemia and reticulocytopenia, and died on post-natal day 10 (P10) due to bone marrow failure without stress erythropoiesis in the spleen. These mice exhibited a progressive exhaustion of the hematopoietic stem and progenitor cell (HSPC) compartment by E17.5, resulting in a 40% reduction in the number of erythroid cells at the BFU-E stage. Flow cytometry analyses of terminal erythroid differentiation further demonstrated a 30% reduction in the number of basophilic erythroblasts at E17.5. In contrast with Vav-iCre;Rps19fl/+ mice, Vav-iCre;Rpl5fl/+ mice died perinatally from severe anemia. They also differed markedly from Rps19 haplo-insufficient mice as they exhibited a progressive expansion of the HSPC compartment. Rather, the defect in Rpl5 haplo-insufficient mice was restricted to the erythropoietic compartment, with an accumulation of BFU-E and CFU-E erythroid progenitors and a 70% decrease in the number of basophilic erythroblasts by E17.5. To elucidate the mechanisms underlying the specific HSPC phenotypes in Rps19 and Rpl5 haplo-insufficient mice, we performed scRNAseq analyses of fetal liver cells at E13.5. While Vav-iCre;Rps19fl/+ mice presented global alterations of the transcriptome at each stage of differentiation, Vav-iCre;Rpl5fl/+ mice only presented defects in erythroid lineage cells, beginning at the proerythroblast stage. Comparative analyses revealed Uba52, encoding the core RPL40 ribosomal protein, as a differentially regulated gene and western blot analyses confirmed decreased expression of RPL40 in both models, albeit to different extents. RPL40 is associated with translation elongation, and accordingly, we observed acceleration of translation by polysome profiling in the Rps19 model, along with decreases in the phosphorylation of the elongation factor eEF2 in the ckit+ population, corroborating the polysome profiling results. By E17.5, the phosphorylation of both the translation initiation factor eIF2a and elongation factor eEF2 were reduced, indicative of increased translation in the mutant mice. Accordingly, we observed reduced mTOR signaling in both these models. Furthermore, Vav-iCre;Rps19fl/+ fetal livers demonstrated activation of the p53 pathway, consistent with clinical findings. However, to our surprise, expression of Runx1, a transcription factor that mediates ribosome biogenesis, was significantly increased. Importantly, activation of both p53 and Runx1 pathways played critical roles in the pathological hematopoiesis occurring in Rps19 haplo-insufficient mice as erythroid defects were rescued by conditional deletion of either p53 or Runx1. Deletion of one or both Runx1 alleles significantly rescued fetal HSPC defects but bi-allelic p53 loss was needed to fully rescue fetal HSC exhaustion and prevent neonatal lethality in Vav-iCre;Rps19fl/+ mice. Taken together, our results unravel distinct requirements for Rps19 and Rpl5 during fetal hematopoiesis and provide novel insights into the mechanism(s) behind ribosomal protein haploinsufficiency leading to DBA.

Diamond Blackfan贫血（DBA）是一种先天性骨髓衰竭综合征，通常与核糖体基因的突变或缺失有关，导致蛋白质单倍体不足，进而引起核仁应激和p53激活。临床上，DBA通常在出生后表现出来，其特征为大红细胞症、网织红细胞减少以及骨髓中红系前体细胞缺乏。患者还可能在其他造血谱系中出现额外的缺陷，这表明造血干细胞和/或祖细胞存在缺陷。然而，值得注意的是，目前仍不清楚这些缺陷是所有核糖体蛋白（RP）突变所共有的，还是针对大核糖体亚基与小核糖体亚基有所不同。此外，由于缺乏与核糖体病临床相关的小鼠模型，人们对核糖体蛋白在胎儿造血过程中的作用了解甚少。为了解决这些问题，我们利用CRISPR/Cas9技术构建了Rps19^{fl/fl}和Rpl5^{fl/fl}条件性小鼠模型——代表DBA中最常见的突变基因型，并将它们与Vav - iCre小鼠进行杂交。Vav - iCre;Rps19^{fl/+}小鼠重现了DBA的临床特征，包括大细胞性贫血和网织红细胞减少，并在出生后第10天（P10）因骨髓衰竭死亡，且脾脏中无应激性红细胞生成。这些小鼠在E17.5时造血干细胞和祖细胞（HSPC）区室逐渐耗竭，导致BFU - E阶段的红细胞数量减少40%。对终末红细胞分化的流式细胞术分析进一步表明，在E17.5时嗜碱性成红细胞数量减少30%。与Vav - iCre;Rps19^{fl/+}小鼠相反，Vav - iCre;Rpl5^{fl/+}小鼠在围产期因严重贫血死亡。它们也与Rps19单倍体不足小鼠有显著差异，因为它们表现出HSPC区室的逐渐扩张。相反，Rpl5单倍体不足小鼠的缺陷仅限于红细胞生成区室，在E17.5时BFU - E和CFU - E红细胞祖细胞积累，嗜碱性成红细胞数量减少70%。为了阐明Rps19和Rpl5单倍体不足小鼠中特定HSPC表型的潜在机制，我们在E13.5时对胎肝细胞进行了单细胞RNA测序（scRNAseq）分析。虽然Vav - iCre;Rps19^{fl/+}小鼠在分化的每个阶段都呈现出转录组的全局改变，但Vav - iCre;Rpl5^{fl/+}小鼠仅在红系谱系细胞中从原红细胞阶段开始出现缺陷。比较分析显示，编码核心RPL40核糖体蛋白的Uba52是一个差异调节基因，蛋白质印迹分析证实两种模型中RPL40的表达均降低，尽管程度不同。RPL40与翻译延伸有关，相应地，我们通过多聚核糖体图谱分析观察到Rps19模型中翻译加速，同时在ckit⁺群体中延伸因子eEF2的磷酸化降低，这证实了多聚核糖体图谱分析的结果。到E17.5时，翻译起始因子eIF2a和延伸因子eEF2的磷酸化均降低，表明突变小鼠中翻译增加。相应地，我们在这两种模型中都观察到mTOR信号减弱。此外，Vav - iCre;Rps19^{fl/+}胎肝表现出p53通路的激活，这与临床发现一致。然而，令我们惊讶的是，介导核糖体生物发生的转录因子Runx1的表达显著增加。重要的是，p53和Runx1通路的激活在Rps19单倍体不足小鼠发生的病理性造血过程中都起着关键作用，因为通过条件性删除p53或Runx1可挽救红系缺陷。删除一个或两个Runx1等位基因可显著挽救胎儿HSPC缺陷，但需要双等位基因p53缺失才能完全挽救胎儿HSC耗竭并防止Vav - iCre;Rps19^{fl/+}小鼠的新生儿死亡。综上所述，我们的结果揭示了胎儿造血过程中对Rps19和Rpl5的不同需求，并为核糖体蛋白单倍体不足导致DBA的机制提供了新的见解。