Transfer RNAs in Hematopoietic Stem Cell Function

造血干细胞功能中的转移 RNA

• 批准号: 10735318
• 负责人: SUSAN L ACKERMAN
• 金额: $ 31.6万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 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 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10735318
• 关键词: Adult; Age; Amino Acids; Anemia; Anticodon; Arginine; Arginine Specific tRNA; Biological Assay; Blood; Blood Cells; Bone Marrow; Bone marrow failure; Buffers; Cell Count; Cell Differentiation process; Cell Maintenance; Cell physiology; Cells; ChIP-seq; Codon Nucleotides; Data; Defect; Development; Disease; Epitopes; Eukaryota; Failure; Family; Flow Cytometry; Gene Expression; Gene Mutation; Genes; Genetic; Genetic Transcription; Grant; Health; Hematological Disease; Hematopoiesis; Hematopoietic; Hematopoietic Neoplasms; Hematopoietic System; Hematopoietic stem cells; Hemorrhage; Heterogeneity; Housekeeping Gene; Human; Immune; Immunity; Immunologics; Impairment; Individual; Knock-in Mouse; Lead; Life; Maintenance; Malignant - descriptor; Malignant Neoplasms; Messenger RNA; Multigene Family; Mus; Mutation; Natural regeneration; Nature; Neonatal; Neurons; Non-Malignant; Nuclear; Pancytopenia; Phenotype; Population; Production; Proliferating; Protein Biosynthesis; Proteins; Puromycin; RNA; RNA Polymerase III; Regulation; Ribosomal RNA; Ribosomes; Role; Signal Pathway; Stress; Testing; Tissues; Transfer RNA; Translations; Untranslated RNA; Work; analog; cell type; chromatin immunoprecipitation; differential expression; fetal; fetal stem cell; fitness; hematopoietic stem cell self-renewal; leukemia; mRNA Translation; mammalian genome; member; novel; polypeptide; progenitor; programs; reconstitution; response; ribosome profiling; self-renewal; stem cell function; stem cell homeostasis; stem cell self renewal; stem cells; transcriptome; transcriptome sequencing; virtual; young adult

ABSTRACT Hematopoietic Stem Cells (HSCs) produce all cells of the blood lineage throughout life. Defects in HSC self- renewal can lead to immunological defects, anemia, and bone marrow failure. Enhanced HSC self-renewal can result in hematopoietic malignancies. Thus, precise regulation of HSC self-renewal is essential for maintaining hematopoietic and human health. Our previous work has shown that adult HSCs tightly control protein synthesis and that modest changes in protein synthesis impair HSC self-renewal and function. However, the mechanisms that regulate mRNA translation in HSCs remain largely unknown. Transfer RNAs (tRNAs) are non-coding adaptor RNAs critical for mRNA translation that are encoded by hundreds of genes in the mammalian genome, with multiple functional genes capable of decoding virtually every codon. We previously showed that the tRNA repertoire influences neuronal function but the effect of changes in tRNA expression on hematopoietic cells is unknown. In preliminary studies, we found that loss of n-Tr22, a member of the five gene arginine UCU tRNA family significantly impairs HSC maintenance and self-renewal, and this is exacerbated in a sensitized genetic background lacking the ribosome rescue factor Gtpbp2, resulting in a complete loss of adult, but not fetal HSCs. We hypothesize that the sensitivity of adult HSCs to the n-Tr22 mutation may be due to differences in the tRNA repertoire between adult HSCs and restricted progenitors, and between HSCs at different developmental stages. The impact of tRNA mutations may be further influenced by differential codon usage in the transcriptome of these cell populations. Finally, there may be cell-type-specific differences in the signaling pathways activated by loss of a tRNA. We propose to test this hypothesis by using chromatin immunoprecipitation and sequencing to determine the tRNA repertoire in the hematopoietic system. We will also analyze how this tRNA mutation influences the maintenance and function of HSCs in the presence and absence of the Gtpbp2 mutation using flow cytometry and long-term multilineage reconstitution assays. Finally, we will determine the effects of the loss of n-Tr22 and Gtpbp2 on protein synthesis and gene expression by incorporation of a puromycin analog, ribosome profiling, and RNA-sequencing. The results from this grant will not only shed light on the role of tRNAs in regulating mRNA translation in the hematopoietic system, but also provide a means to understand the role of these genes in the phenotypic heterogeneity common to many human hematopoietic disorders.

抽象的 造血干细胞（HSC）在一生中产生血统的所有细胞。 HSC 自身缺陷 更新可能导致免疫缺陷、贫血和骨髓衰竭。增强的 HSC 自我更新能力 导致造血系统恶性肿瘤。因此，HSC自我更新的精确调控对于维持 造血和人体健康。我们之前的工作表明，成体造血干细胞严格控制蛋白质合成 蛋白质合成的适度变化会损害 HSC 的自我更新和功能。然而，这些机制 调节 HSC 中 mRNA 翻译的机制仍然很大程度上未知。转移 RNA (tRNA) 是非编码的 对于 mRNA 翻译至关重要的接头 RNA，由哺乳动物基因组中的数百个基因编码， 具有能够解码几乎每个密码子的多个功能基因。我们之前表明 tRNA 指令集影响神经元功能，但 tRNA 表达变化对造血细胞的影响是 未知。在初步研究中，我们发现n-Tr22（五基因精氨酸UCU tRNA的成员）的缺失 家庭成员显着损害 HSC 的维持和自我更新，而这种情况在敏感的遗传基因中会加剧。 背景缺乏核糖体救援因子 Gtpbp2，导致成人 HSC 完全丧失，但胎儿 HSC 没有完全丧失。 我们假设成人 HSC 对 n-Tr22 突变的敏感性可能是由于 tRNA 的差异 成年 HSC 和受限祖细胞之间以及不同发育阶段的 HSC 之间的库。 tRNA 突变的影响可能进一步受到这些转录组中密码子使用差异的影响。 细胞群。最后，丢失激活的信号通路可能存在细胞类型特异性差异 tRNA 的。我们建议通过使用染色质免疫沉淀和测序来检验这一假设 确定造血系统中的 tRNA 库。我们还将分析这个 tRNA 突变是如何发生的 在存在和不存在 Gtpbp2 突变的情况下影响 HSC 的维持和功能 流式细胞术和长期多谱系重建测定。最后，我们将确定损失的影响 通过掺入嘌呤霉素类似物，n-Tr22 和 Gtpbp2 对蛋白质合成和基因表达的影响， 核糖体分析和 RNA 测序。这笔资助的结果不仅将揭示 tRNA 的作用 调节造血系统中 mRNA 翻译的过程，还提供了一种了解 mRNA 翻译作用的方法 这些基因的表型异质性常见于许多人类造血系统疾病。