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: 10362703
• 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/10362703
• 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; Persons; 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; 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.

摘要 动质体寄生虫布氏锥虫、克氏锥虫和利什曼原虫。影响数百万人 导致人类非洲锥虫病(HAT)、恰加斯病和各种形式的 利什曼病。由于治疗这些被忽视的热带病的药物有限，往往有毒且困难 由于寄生虫对现有药物的耐药性正在上升，因此开发新的化疗药物非常重要。 治疗策略。我们的研究集中在锥虫基因的表达上，因为潜在的 这些机制与人类宿主的机制有很大的不同。例如，多顺反子转录的 蛋白质编码基因和通过剪接前导反式剪接处理前-mRNA是寄生虫特有的步骤 信使核糖核酸合成和成熟。我们在布氏毛滴虫中发现，细胞周期蛋白依赖性激酶的活性 (CDK)反式剪接需要CRK9。通过产生表达模拟敏感的CRK9和 如果没有野生型酶，我们可以在培养细胞中以特定的方式化学抑制该酶。令人惊讶的是， 我们观察到在应用抑制剂后立即发生剪接阻断，这表明CRK9执行必要的 RNA加工机械上的可逆磷酸化。我们的初步数据显示，中国动车组的S之一 底物是SR蛋白和已知的剪接因子TSR1，阻断CRK9活性会影响组装 在剪接体中，执行剪接反应的大而动态的RNA-蛋白质复合体。 因此，我们将确定CRK9如何帮助或控制剪接过程的机制。此外， CDK代表一种高度可药物利用的酶类，而CRK9形成一种不寻常的三聚体酶复合体，与一种 异常的L型细胞周期蛋白和动质体特异性蛋白，表明CRK9是一个有希望的靶点 化疗干预。因此，我们建议对酶复合体进行表征，并确定 最小的复合体是活跃的，可以作为未来高吞吐量的先决条件进行重组表达 抑制剂筛选。最后，基于初步数据，我们将检验CRK9是 化合物SCYX-7158，目前正在进行抗HAT的临床试验。