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.

项目总结/摘要 核糖体病包括一系列遗传异常导致核糖体受损的疾病 生物起源和功能。尽管所有类型的细胞都需要核糖体，但核糖体病显示出特异性的clin-DNA结合。 临床表型人孤立性先天性无脾（伊卡），在出生时表现为脾缺失， 或作为发育不全的脾脏，没有额外的发育异常，最近已被归类为一个ri- 乳房病鉴于脾脏在宿主防御中的核心作用，伊卡患者很容易受到生命威胁- 威胁着血液传播的感染在基因编码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组分]在大部分伊卡pa中， tients。RPSA在人类脾脏发育中的关键作用是令人惊讶的，考虑到其他基因突变， RP从未与无脾相关。而脾的发育是一个理想的研究系统 由于RP的特定功能，迄今为止产生的动物模型不能用于确定 RPSA影响脾脏发育。实际上，无效Rpsa等位基因的纯合性导致子宫内的早期致死， 而杂合性与无脾无关。为了克服这些局限性， RPSA脾特异性功能，我们已经产生了小鼠胚胎脾细胞缺乏， Rpsa或Rps 14（值得注意的是，Rps 14的单倍不足导致人类红细胞生成受损，而不影响红细胞生成）。 脾缺陷）。初步结果表明，细胞增殖和蛋白质合成更显着地恢复， 在Rpsa与Rps 14突变脾细胞系中诱导。然而，人们对这种现象背后的机制知之甚少。 小鼠脾细胞中RPSA依赖性表型的特异性以及这些表型的潜在保守性 人类的机制。我们假设RPSA发挥“专门”的作用，仅限于脾脏 在小鼠和人类中的发展。我们计划通过这些具体目标来测试这个假设：1。评估 在易处理小鼠脾模型系统中RPSA单倍不足特异性。通过使用无偏 全基因组方法（Ribo-seq），我们将评估翻译动力学，我们将积极测量trans- 野生型和突变型（分别缺乏Rpsa和Rps 14）小鼠胚胎脾中的lated mRNA 间充质细胞这些实验将定义RPSA依赖性机制和翻译靶点， 使脾胚胎细胞相对于RPS 14更容易丧失RPSA。2.评估RPSA是否 功能在人脾模型系统中是保守的。我们将生成并描述人类 通过在脾间充质细胞中进行RPSA的基因组编辑， 从人类胎儿中获得的细胞。这些实验将确定是否RPSA依赖的缺陷， 在Aim 1中鉴定的小鼠胚胎脾细胞在人胎儿脾细胞中是保守的。这项研究将 具有开创性，因为它将揭示支撑核糖体蛋白质特定功能的独特机制 在脾脏发育，以及揭示伊卡的发病机制，危及生命的出生缺陷。