Function and regulation of the essential RNA binding protein, DRBD18

必需 RNA 结合蛋白 DRBD18 的功能和调节

• 批准号: 10649589
• 负责人: Laurie K. Read
• 金额: $ 41.46万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-06-06 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10649589
• 关键词: 3' Untranslated Regions; 5' Untranslated Regions; Affect; Africa South of the Sahara; African Trypanosomiasis; Apical; Arginine; Binding; Binding Proteins; Biochemical; Biological; Biological Assay; Biological Process; Biology; Biotinylation; Blood Circulation; Cell physiology; Cytoplasm; Data; Development; Disease; Drug Targeting; Event; Gene Expression; Gene Expression Regulation; Genetic; Genomics; Goals; Homeostasis; Human; Immunoprecipitation; Individual; Insect Vectors; Insecta; Kinetoplastida; Knowledge; Laboratories; Lead; Livestock; Mass Spectrum Analysis; Mastigophora; Mediating; Medical; Messenger RNA; Methylation; Modeling; Molecular; Morbidity - disease rate; Nuclear; Nuclear Export; Nucleotides; Oligonucleotides; Open Reading Frames; Organism; Parasites; Pathway interactions; Pattern; Peptide Initiation Factors; Pharmaceutical Preparations; Population; Positioning Attribute; Post-Transcriptional Regulation; Post-Translational Protein Processing; Process; Protein Binding Domain; Protein Kinase; Proteins; Proteomics; RNA; RNA-Binding Proteins; Regulation; Regulator Genes; Regulatory Pathway; Research; Resolution; Role; Specificity; Testing; Transcript; Translation Initiation; Translations; Trypanosoma; Trypanosoma brucei brucei; Vaccination; Variant; Virulence; cohort; combinatorial; crosslink; experimental study; genome-wide; human mortality; in vivo; insight; mRNA Export; mRNA Stability; mRNA Transcript Degradation; mRNA Translation; posttranscriptional; protein function; protein protein interaction; ribosome profiling; transcriptome sequencing; translational impact

ABSTRACT The flagellated protozoan, Trypanosoma brucei, is a devastating human and veterinary parasite in sub- Saharan Africa, and the causative agent of Human African Trypanosomiasis (HAT). HAT is fatal if untreated, vaccination is not an option, and available drugs are toxic, difficult to administer, and expensive. In the search for new treatments, understanding the basic biology of the parasite is a cornerstone on the path to discovery of unique biological processes that could potentially serve as drug targets. Trypanosomes are exceptional in that they perform gene regulation almost exclusively at posttranscriptional levels, through control of processes such as mRNA stability and translational efficiency. This reliance on posttranscriptional regulation necessitates that RNA binding proteins (RBPs) are the key effectors of trypanosome development, homeostasis, and virulence. Our laboratory discovered DRBD18, an abundant RBP that is essential for the survival of both the human bloodstream form (BF) and the insect vector procyclic form (PF) of T. brucei, and that is not conserved outside the Order Kinetoplastida. DRBD18 depletion in the PF results in significant changes in the abundance of nearly 1000 mRNAs, many of the most highly regulated themselves encoding RBPs and protein kinases. Thus, DRBD18 is positioned at the apex of numerous potential regulatory cascades. Proteomic data suggest that DRBD18 functions in both nuclear mRNA export and translation initiation. Remarkably, the ability of DRBD18 to stabilize or destabilize mRNAs as well as both its protein and mRNA binding specificities are dramatically regulated by arginine methylation, which acts as a molecular switch towards DRBD18 action. In the proposed studies, we will elucidate DRBD18 functions and regulation by 1) identifying direct DRBD18 mRNA targets, 2) mechanistically defining DRBD18 effector pathways, and 3) establishing the methylation-responsiveness of DRBD18 interactions and DRBD18 functions. In Aim 1, we will use iCLIP to define on a genome-wide level the sets of mRNAs that are directly bound by DRBD18 and to determine how these sets of bound mRNAs are regulated upon DRBD18 methylation. In Aim 2, we will determine the roles of DRBD18 in nuclear mRNA export and translation initiation, and test hypotheses regarding regulation of these functions by arginine methylation. In Aim 3, we will study combinatorial RBP interactions and test the hypothesis that methylation-sensitive interactions between DRBD18 and other RBPs regulate the specificity of DRBD18 mRNA targeting. Our studies will define distinct cis-trans modules that mediate methylation-sensitive DRDB18 functions. Using combined genetic, genomic, and biochemical approaches, the proposed studies will provide fundamental insights into specific gene regulatory events in T. brucei, and uncover regulatory mechanisms with wide- ranging applicability in trypanosomes. They also have the potential to broaden our understanding of RNA biology and its regulation by arginine methylation in higher organisms.

抽象的 鞭毛的原生动物Brucei是毁灭性的人类和兽医寄生虫 撒哈拉非洲和人类非洲锥虫病的病因（HAT）。帽子是致命的，如果没有治疗， 疫苗接种不是一种选择，可用的药物有毒，难以给药且昂贵。在搜索中 对于新疗法，了解寄生虫的基本生物学是发现途径的基石 可能有可能用作药物靶标的独特生物学过程。锥虫是例外的 他们通过控制此类过程，几乎完全在转录后级别执行基因调控 作为mRNA稳定性和翻译效率。对转录后法规的这种依赖，需要 RNA结合蛋白（RBP）是锥虫发育，稳态和毒力的关键效应因子。 我们的实验室发现了DRBD18，这是一种对人类生存至关重要的丰富的RBP Brucei的血液形式（BF）和昆虫矢量procyclic形式（PF），这不是外部保守的 订单动力层。 PF的DRBD18耗竭导致了几乎的丰度变化 1000个mRNA，许多最受高度调节的人，编码RBP和蛋白激酶。因此， DRBD18位于众多潜在调节级联的顶点。蛋白质组学数据表明 DRBD18在核mRNA输出和翻译启动中起作用。值得注意的是，DRBD18的能力 稳定或破坏mRNA及其蛋白质和mRNA结合的特异性都显着 由精氨酸甲基化调节，该甲基化是向DRBD18作用的分子转换。在提议中 研究，我们将通过1）识别直接DRBD18 mRNA靶标阐明DRBD18的功能和调节，2） 从机械上定义DRBD18效应器途径，3）建立甲基化反应性 DRBD18相互作用和DRBD18功能。在AIM 1中，我们将使用ICLIP在全基因组水平上定义 直接受DRBD18绑定并确定这些绑定的mRNA的mRNA集 根据DRBD18甲基化调节。在AIM 2中，我们将确定DRBD18在核mRNA输出中的作用 和翻译起始，以及关于通过精氨酸甲基化调节这些功能的假设。 在AIM 3中，我们将研究组合RBP相互作用并检验甲基化敏感的假设 DRBD18与其他RBP之间的相互作用调节DRBD18 mRNA靶向的特异性。我们的 研究将定义介导对甲基化敏感的DRDB18功能的不同的顺式传播模块。使用 联合遗传，基因组和生化方法，拟议的研究将提供基本的研究 洞察布鲁氏菌中特定基因调节事件的见解，并发现具有广泛的调节机制 在锥虫中的适用性。他们也有潜力扩大我们对RNA的理解 生物学及其对高等生物中精氨酸甲基化的调节。

项目成果

Selective nuclear export of mRNAs is promoted by DRBD18 in Trypanosoma brucei.

• DOI: 10.1111/mmi.14773
• 发表时间: 2021-09
• 期刊: Molecular microbiology
• 影响因子: 3.6
• 作者: Mishra A;Kaur JN;McSkimming DI;Hegedűsová E;Dubey AP;Ciganda M;Paris Z;Read LK
• 通讯作者: Read LK

Translational control by Trypanosoma brucei DRBD18 contributes to the maintenance of the procyclic state.

• DOI: 10.1261/rna.079625.123
• 发表时间: 2023-12
• 期刊: RNA (New York, N.Y.)