Catalytic roles of RNA methyltransferase DIMT1

RNA甲基转移酶DIMT1的催化作用

• 批准号: 10522085
• 负责人: BARRY S. COOPERMAN
• 金额: $ 60.19万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10522085
• 关键词: Ablation; Affect; Basic Science; Biogenesis; Biological; Biological Assay; Cell Line; Cell Survival; Cells; Complex; DNA Damage; DNA Repair; DNA Repair Pathway; Data; Development; Discipline; Disease; Enzymes; Event; Fanconi' s Anemia; Gene Expression; Genetic Translation; Goals; Hematopoiesis; Hematopoietic; Hematopoietic stem cells; Human; Human Pathology; Impairment; In Vitro; Internal Ribosome Entry Site; Kinetics; Knowledge; Laboratories; Mediating; Messenger RNA; Methylation; Methyltransferase; Modification; Molecular; Pathologic Processes; Pathway interactions; Peptide Initiation Factors; Peptides; Physiological Processes; Physiology; Process; Protein Biosynthesis; RNA; Reaction; Regulation; Reproducibility; Research; Ribosomal Frameshifting; Ribosomal RNA; Ribosomes; Role; Site; Specificity; Structure; Structure-Activity Relationship; System; Transfer RNA; Translations; Variant; base; cell type; cricket paralysis virus; insight; mRNA Decay; novel; novel therapeutics; polypeptide; response; scaffold; success

Ribosome RNA (rRNA) modifying enzymes are important factors in the assembly and function of ribosomes, dysregulation of which is frequently seen in human hematopoietic disorders. We only have limited knowledge of the catalytic role of rRNA modifying enzymes in the physiological and pathological processes of human hematopoiesis, although a few studies characterized the importance of rRNA modifications in these processes. Preliminary data (CRISPR screening) from my laboratory shows that the ablation of DIMT1 (an 18S rRNA Ni·6A-dimethylation (mi·6A) methyltransferase) significantly impairs cell viability of hematopoietic stem progenitor cells (HSPCs). DIMT1 is highly expressed in HSPC, and its expression decreases in the differentiated lineages. However, the molecular function and detailed mechanism of DIMT1 in human hematopoiesis remain elusive. Even though DIMT1-mediated rRNA modifications are highly conserved, the catalytic activity of DIMT1 is dispensable for proper ribosome biogenesis. However, our recent results suggest that the catalytic activity of DIMT1 is required for the viability of HSPCs, while the catalytic-independent role of DIMT1 is important for 18S rRNA processing and ribosome biogenesis. This implies that DIMT1 may have a necessary role as a scaffold in ribosome biogenesis separate from its catalytic activity. Furthermore, we show that the catalytic role of DIMT1 is indispensable for the expression of genes involved in the Fanconi anemia and other DNA repair pathways. However, whether and how DIMT1-mediated mi·6A installation in 18S rRNA influences cell viability and DNA repair pathways in HSPCs are not known. Here, our goal is to understand the molecular function and the mechanistic details of the catalytic role of DIMT1 in the regulation of cell viability and gene expression in HSPCs. Specifically, Aim 1 will investigate the catalytic role of DIMT1 as an 18S rRNA methyltransferase in HSPC cell viability and protein synthesis. Aim 2 will investigate the molecular mechanism by which DIMT1- mediated m26•6A in 18S rRNA controls translation elongation using an in vitro eukaryotic translation system. Aim 3 will investigate the structure-function relationships and catalytic mechanism of DIMT1. Results from these studies will provide novel insights into how the catalytic role of DIMT1 impacts HSPC cell viability and the expression of genes Fanconi anemia and other DNA repair pathways. The understanding we will gain can be broadly applicable to hematopoietic studies and many other biological disciplines.

核糖体RNA(RRNA)修饰酶是影响核糖体RNA的组装和功能的重要因素 核糖体，常见于人类造血系统疾病中。我们只有有限的 了解rRNA修饰酶在急性呼吸窘迫综合征的生理和病理过程中的催化作用 人类造血，尽管少数研究表明rRNA修饰在这些疾病中的重要性 流程。来自我的实验室的初步数据(CRISPR筛查)表明，DIMT1(An 18s)的消融 RRNA Ni·6A-二甲基化(mI·6A)甲基转移酶对造血干细胞活性的影响 祖细胞(HSPC)。DIMT1在HSPC中高表达，在分化的HSPC中低表达 血统。然而，DIMT1在人类造血中的分子功能和详细机制仍然存在。 难以捉摸。尽管DIMT1介导的rRNA修饰是高度保守的，但其催化活性 DIMT1对于正常的核糖体生物发生是必不可少的。然而，我们最近的结果表明，催化剂 DIMT1的活性是HSPC存活所必需的，而DIMT1的非催化作用是重要的 用于18S rRNA加工和核糖体生物发生。这意味着DIMT1可能具有必要的角色 核糖体生物发生中的支架与其催化活性是分开的。此外，我们还证明了它的催化作用 DIMT1对于参与Fanconi贫血和其他DNA修复的基因的表达是必不可少的 小路。然而，DIMT1介导的MI·6A安装在18S rRNA中是否以及如何影响细胞活力 HSPC中的DNA修复途径尚不清楚。在这里，我们的目标是了解分子功能和 DIMT1在细胞活力和基因表达调控中的催化作用的机制细节 HSPC。具体地说，Aim 1将研究DIMT1作为18S rRNA甲基转移酶在 HSPC细胞活力和蛋白质合成。Aim 2将研究DIMT1- 18S rRNA中介导的M26·6A利用体外真核翻译系统控制翻译延伸。目标 3研究DIMT1的构效关系和催化机制。来自这些的结果 研究将为DIMT1的催化作用如何影响HSPC细胞活性和 范可尼贫血等DNA修复途径的基因表达。我们将获得的理解可以是 广泛适用于造血学研究和许多其他生物学学科。