The essential role of cyclin-dependent kinase CRK9 in trypanosome pre-mRNA processing

细胞周期蛋白依赖性激酶 CRK9 在锥虫前 mRNA 加工中的重要作用

• 批准号: 10219576
• 负责人: ARTHUR GUNZL
• 金额: $ 41万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-03-02 至 2026-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10219576
• 关键词: Ablation; Affect; African Trypanosomiasis; Alanine; Animals; Architecture; Binding; Blood Circulation; Cell Cycle; Cell Cycle Completion; Cell Death; Cell Line; Cells; Chagas Disease; Chemicals; Clinical; Clinical Trials; Code; Complex; Cultured Cells; Cutaneous; Cyclin-Dependent Kinases; Cyclins; Cytokinesis; Data; Defect; Developing Countries; Development; Disease; Docking; Drug Targeting; Drug resistance; Enzymes; Future; Gene Expression; Genes; Genetic Transcription; Germ; Human; Immunoprecipitation; In Vitro; Individual; Infection; Intervention; Leishmania; Leishmaniasis; Libraries; Link; Messenger RNA; Mitosis; Multienzyme Complexes; Mus; Mutate; Mutation; Nuclear; Parasite resistance; Parasites; Pharmaceutical Preparations; Phase II/III Trial; Phosphorylation; Phosphotransferases; Polyadenylation; Pre-mRNA Polyadenylation Factor; Process; Proteins; RNA; RNA Processing; RNA Splicing; Reaction; Recombinants; Research; Role; Signal Pathway; Site; Spliced Leader RNA; Spliced Leader Sequences; Spliceosome Assembly Pathway; Spliceosomes; Structure; System; Technology; Testing; Trans-Splicing; Translations; Trypanosoma; Trypanosoma brucei brucei; Trypanosoma cruzi; Trypanosomiasis; Visceral; Wheat; analog; base; deviant; genome-wide; high throughput screening; human disease; inhibitor/antagonist; mRNA Precursor; neglected tropical diseases; overexpression; protein complex; small molecule inhibitor; tool; transcriptome sequencing

Summary The kinetoplastid parasites Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. affect millions of people worldwide, causing Human African Trypanosomiasis (HAT), Chagas’ disease, and various forms of Leishmaniasis, respectively. Since drugs for these neglected tropical diseases are limited, often toxic and difficult to administer, and parasite resistance to existing drugs is on the rise, it is important to develop new chemo- therapeutic strategies. Our research is focused on trypanosome gene expression because the underlying mechanisms deviate substantially from those in the human host. For example, polycistronic transcription of protein coding genes and processing of pre-mRNA by spliced leader trans splicing are parasite-specific steps in mRNA synthesis and maturation. We discovered in T. brucei that the activity of the cyclin-dependent kinase (CDK) CRK9 is required for trans splicing. By generating a cell line that expresses analog-sensitive CRK9 and no wild-type enzyme, we could chemically inhibit the enzyme in specific manner in cultured cells. Surprisingly, we observed an instant splicing block after applying the inhibitor, suggesting that CRK9 carries out essential reversible phosphorylation on the RNA processing machinery. Our preliminary data indicate that one of CRK9’s substrate is the SR protein and known splicing factor TSR1, and that blocking CRK9 activity affects the assembly of the spliceosome, the large and dynamic RNA-protein complex that carries out the splicing reaction. Consequently, we will determine the mechanism of how CRK9 aids or controls the splicing process. Furthermore, CDKs represent a highly druggable enzyme class, and CRK9 forms an unusual trimeric enzyme complex with a deviant L-type cyclin and a kinetoplastid-specific protein, suggesting that CRK9 is a promising target for chemotherapeutic intervention. Therefore, we propose to characterize the enzyme complex and determine a minimal complex that is active and can be expressed recombinantly as prerequisite for future high throughput inhibitor screens. Finally, based on preliminary data, we will test the hypothesis that CRK9 is the target of the compound SCYX-7158 which is currently in clinical trials against HAT.

概括 动型寄生虫Brucei，Cruzi和Leishmania spp。影响数百万 在全球范围内，引起非洲人类锥虫病（帽子），夏加斯病和各种形式的人类 利什曼病分别。由于这些被忽视的热带疾病的药物受到限制，通常有毒且困难 进行管理和对现有药物的抗寄生虫的抗性正在上升，开发新的化学疗法很重要 治疗策略。我们的研究重点是锥体基因表达 机制大大偏离了人类宿主中的机制。例如，多元转录的 蛋白质编码基因和通过剪接的Leader Trans剪接对MRNA的处理是寄生虫特异性的步骤 mRNA合成和成熟。我们在T. brucei中发现细胞周期蛋白依赖性激酶的活性 （CDK）CRK9是反剪接所必需的。通过生成表达对模拟敏感CRK9的细胞系和 没有野生型酶，我们可以在培养细胞中以特定方式化学抑制酶。出奇， 我们观察到施加抑制剂后的即时剪接块，表明CRK9进行了必不可少的 RNA加工机械上的可逆磷酸化。我们的初步数据表明CRK9之一 底物是SR蛋白和已知的剪接因子TSR1，并且阻断CRK9活性会影响组装 在剪接体中，进行剪接反应的大型且动态的RNA蛋白质复合物。 因此，我们将确定CRK9如何辅助或控制剪接过程的机制。此外， CDK代表高度可拖动的酶类，CRK9与A形成不寻常的三聚酶复合物 异常的L型细胞周期蛋白和动力质体特异性蛋白质，这表明CRK9是对 化学治疗干预。因此，我们建议表征酶复合物并确定A 最小的复合物，可以重组表达为未来高通量的先决条件 抑制剂屏幕。最后，基于初步数据，我们将检验以下假设：CRK9是该目标 目前正在针对HAT的临床试验中，复合SCYX-7158。