Specificity of RPSA-dependent translational control in mouse and human fetal spleen cells

小鼠和人胎儿脾细胞中 RPSA 依赖性翻译控制的特异性

• 批准号: 10647605
• 负责人: Maurizio Risolino
• 金额: $ 16.15万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10647605
• 关键词: Address; Affect; Alleles; Animal Model; Bacteria; Biogenesis; Biological Models; Biology; Birth; Bone Marrow; Cartilage-hair hypoplasia; Cell Culture System; Cell Culture Techniques; Cell Line; Cell Proliferation; Cells; Child; Classification; Clustered Regularly Interspaced Short Palindromic Repeats; Collection; Comprehension; Congenital Abnormality; Defect; Development; Developmental Process; Diamond; Disease; Dyskeratosis Congenita; Dysmyelopoietic Syndromes; Embryo; Encapsulated; Erythropoiesis; Fetal Spleen; Fetus; Genes; Heterozygote; Host Defense; Human; Impairment; Individual; Infection; Knowledge; Life; Light; Link; Literature; Loss of Heterozygosity; Macrocytic Anemia; Mandibulofacial Dysostosis; Measures; Mediating; Mesenchymal; Messenger RNA; Modeling; Molecular; Molecular Abnormality; Mouse Strains; Mus; Mutation; Newborn Infant; Organogenesis; Pathogenesis; Patients; Phenocopy; Phenotype; Play; Predisposition; Process; Protein Biosynthesis; Proteins; Research; Ribosomal Proteins; Ribosomes; Role; Specificity; Spleen; Spleen Development; System; Testing; Tissues; Translating; Translations; blastomere structure; body system; cell type; clinical phenotype; embryo cell; erythroid differentiation; exome sequencing; experimental study; genome editing; genome-wide; in utero; in vivo; loss of function; mouse model; mutant; ribosome profiling; ribosomopathy

Project Summary/Abstract Ribosomopathies comprise a collection of disorders in which genetic abnormalities cause impaired ribosome biogenesis and function. Despite the need for ribosomes in all cell types, ribosomopathies display specific clin- ical phenotypes. Human Isolated Congenital Asplenia (ICA), which manifests at birth as absence of the spleen, or as hypoplastic spleen, without additional developmental anomalies, has been recently classified as a ri- bosomopathy. Given the central role of the spleen in host defense, ICA patients are vulnerable to life- threatening blood-borne infections. Germline heterozygous mutations were identified in the gene encoding RPSA [ribosomal protein (RP) SA; a component of the ribosome small subunit] in a large proportion of ICA pa- tients. The critical role for RPSA in human spleen development was surprising, given that mutations in other RPs had never been associated with asplenia. While spleen development constitutes an ideal system to study specific functions of RPs, animal models generated so far cannot be used to determine how deficiency of RPSA affects spleen development. Indeed, homozygosity for a null Rpsa allele causes early lethality in utero, while heterozygosity is not associated with asplenia. In order to overcome these limitations and to identify RPSA spleen-specific functions, we have generated mouse embryonic spleen cells with deficiency of either Rpsa or Rps14 (of note, haploinsufficiency of RPS14 causes impaired erythropoiesis in humans, without spleen defects). Preliminary results show that cell proliferation and protein synthesis are more strikingly re- duced in Rpsa versus Rps14 mutant splenic cell lines. Yet, little is known about the mechanisms underlying the specificity of RPSA-dependent phenotypes in mouse spleen cells and the potential conservation of these mechanisms in humans. We hypothesize that RPSA plays “specialized” roles that are restricted only to spleen development in mouse and humans. We plan to test this hypothesis via these specific aims: 1. To assess specificity of RPSA haploinsufficiency in a tractable murine spleen model system. By using unbiased genome-wide approaches (Ribo-seq), we will assess translation dynamics and we will measure actively trans- lated mRNAs in wildtype and mutant (deficient for Rpsa and Rps14, respectively) mouse embryonic spleen mesenchymal cells. These experiments will define RPSA-dependent mechanisms and translational targets that render spleen embryonic cells more susceptible to loss of RPSA versus RPS14. 2. To assess whether RPSA functions are conserved in a human spleen model system. We will generate and characterize human splenic cell cultures with RPSA deficiency by performing genome editing of RPSA in splenic mesenchymal cells obtained from human fetuses. These experiments will determine whether RPSA-dependent defects in mouse embryonic spleen cells, identified in Aim1, are conserved in human fetal spleen cells. This research will be ground-breaking as it will uncover unique mechanisms underpinning specific functions of ribosomal proteins in spleen development, as well as shedding light on the pathogenesis of ICA, a life-threatening birth defect.

项目摘要/摘要 核糖体病包括一系列疾病，其中遗传异常导致核糖体受损。 生物发生和功能。尽管所有类型的细胞都需要核糖体，但核糖体疾病表现出特殊的临床表现。 临床表型。人类孤立性先天性无脾(ICA)，出生时表现为无脾， 或为发育不良的脾，没有额外的发育异常，最近被归类为Ri-1。 乳腺病。鉴于脾在宿主防御中的核心作用，ICA患者很容易受到生命的影响- 有血液传播感染的危险。在编码基因中发现了种系杂合突变 RpsA[核糖体蛋白(RP)SA；核糖体小亚基的一种成分]在很大比例的ICA pa- 蒂恩斯。RPSA在人类脾发育中的关键作用令人惊讶，因为其他基因的突变 RPS从未与脾功能不全联系在一起。而脾的发育构成了一个理想的研究体系 RPS的特定功能，到目前为止已经建立的动物模型不能用来确定如何缺乏 RpsA影响脾发育。事实上，rpsA零等位基因的纯合子会导致子宫内的早期死亡， 而杂合性与脾功能不全无关。为了克服这些限制并确定 RpsA脾特异功能，我们产生的小鼠胚胎脾细胞缺乏其中之一 RpsA或Rps14(值得注意的是，RPS14的单倍体不足会导致人类红细胞生成受损，而不是 脾虚)。初步结果显示，细胞增殖和蛋白质合成更显著地恢复 在rpsA和Rps14突变的脾细胞系中诱导表达。然而，人们对其背后的机制知之甚少。 小鼠脾细胞RPSA依赖表型的特异性及其潜在保守性 人类的机制。我们假设RPSA扮演着仅限于脾的“特殊”角色 老鼠和人类的发育。我们计划通过以下具体目标来检验这一假设：1.评估 RPSA单倍体功能不全在易处理的小鼠脾模型系统中的特异性。通过使用不偏不倚 全基因组方法(RIBO-SEQ)，我们将评估翻译动态，我们将积极衡量反式 野生型和突变型(分别缺乏rpsA和Rps14)小鼠胚胎脾中的晚期mRNAs 间充质细胞。这些实验将定义依赖RPSA的机制和翻译靶点 使脾胚胎细胞更容易失去RPSA而不是RPS14。2.评估RPSA是否 在人类脾模型系统中，功能是保守的。我们将生成并刻画人类 在脾间充质中进行RPSA基因组编辑培养RPSA缺乏症的脾细胞 从人类胎儿中获得的细胞。这些实验将确定RPSA依赖的缺陷在 在Aim1中鉴定的小鼠胚胎脾细胞在人胎脾细胞中是保守的。这项研究将 具有开创性，因为它将揭示支撑核糖体蛋白特定功能的独特机制 在脾发育方面的研究，以及揭示ICA的发病机制，ICA是一种威胁生命的出生缺陷。