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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

基本信息

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
项目类别：
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

项目摘要

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.

总结