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）一生都会产生所有血统的细胞。 HSC自我缺陷 更新会导致免疫缺陷，贫血和骨髓衰竭。增强的HSC自我更新可以 导致造血恶性肿瘤。因此，HSC自我更新的精确调节对于维护至关重要 造血和人类健康。我们以前的工作表明，成年HSC紧密控制蛋白质合成 蛋白质合成的适度变化会损害HSC的自我更新和功能。但是，机制 调节HSC中的mRNA翻译在很大程度上仍然未知。转移RNA（TRNA）是非编码的 适配器RNA对mRNA翻译至关重要，mRNA翻译至关重要的哺乳动物基因组中的数百个基因编码， 具有多个功能基因，能够几乎解码每个密码子。我们以前表明tRNA 曲目影响神经元功能，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的丢失是五种基因精氨酸ucu tRNA的成员 家庭大大损害了HSC的维护和自我更新，这在敏感的遗传中加剧了 背景缺乏核糖体救援因子GTPBP2，从而完全损失了成人，但没有胎儿HSC。 我们假设成年HSC对N-TR22突变的敏感性可能是由于tRNA的差异 成人HSC和受限祖细胞之间以及不同发育阶段的HSC之间的曲目。 tRNA突变的影响可能会受到这些转录组中差分密码子使用的进一步影响 细胞种群。最后，在损失激活的信号通路中可能存在细胞类型特异性差异 trna。我们建议通过使用染色质免疫沉淀和测序到 确定造血系统中的tRNA库。我们还将分析该tRNA突变如何 在使用和不存在GTPBP2突变的情况下，HSC的维护和功能 流式细胞仪和长期多核重建测定法。最后，我们将确定损失的影响 N-TR22和GTPBP2在蛋白质合成和基因表达上，通过掺入紫霉素类似物， 核糖体分析和RNA测序。这笔赠款的结果不仅会阐明TRNA的作用 在调节造血系统中的mRNA翻译时，也提供了一种理解作用的方法 这些基因在许多人类造血疾病共有的表型异质性中。