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Structure, regulation, and evolution of the splicing machinery

熔接机械的结构、调节和演变

基本信息

批准号：
10406517
负责人：
Manuel Ares
金额：
$ 51.89万
依托单位：
UNIVERSITY OF CALIFORNIA SANTA CRUZ
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-05-16 至 2027-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10406517
关键词：
Address Alternative Splicing Antibiotics Area Awareness Bacteria Basic Science Biochemical Candidate Disease Gene Cell physiology Cells Complex Defect Disease Drosophila genus Engineering Eukaryota Evolution Fruit Gene Expression Regulation Gene Proteins Genes Genetic Transcription Health Human Individual Intervention Introns Investigation Knowledge Lethal Genes Malignant Neoplasms Measures Mediating Mutation Nature Noise Output Pathway interactions Process RNA Polymerase II RNA Splicing RNA-Binding Proteins Reaction Recurrent Malignant Neoplasm Regulation Reporter Role Site Spinal Muscular Atrophy Spliceosome Assembly Pathway Spliceosomes Structure System Testing Time Translating U2 Small Nuclear Ribonucleoprotein Variant Work Yeasts base cell growth experimental study improved in vivo innovation mRNA Precursor novel novel strategies predictive modeling repaired sex success synthetic biology transcriptome sequencing tumor progression

项目摘要

PROJECT SUMMARY The complexity of human splicing is daunting, yet intervention in splicing for treatment of diseases holds huge potential. Based on strong preliminary results, we propose three areas of investigation that leverage our group’s deep knowledge of splicing to address critical open questions, and to explore the potential for innovative engineering. The first area addresses the mechanism by which U2 snRNP captures the intron branchpoint early in spliceosome assembly, a step altered by recurrent cancer mutations and targeted in nature by antibiotic-producing bacteria. Using new reporters in which two branchpoints compete for recognition, we have identified a novel splicing fidelity mechanism we call “NO-BP decay,” in which U2 complexes that fail due to aberrant branchpoint selection are destroyed. We will characterize this process, applying a battery of candidate gene-based suppressor screens and biochemical tests in splicing extracts. The second area of investigation addresses how splicing is integrated with transcription and cell growth at the individual gene and cellular levels, an emerging area in need of innovation if splicing is to be successfully engineered. Preliminary results indicate that yeast cells have a limited capacity for splicing that creates competition for pre-mRNAs that is critical to cell function. We will measure both splicing capacity and the dynamics of competition, using RNA sequencing to develop a predictive model that explains how splicing is coordinated at a systems level. To understand the contribution of individual genes to this system we are applying synthetic biology approaches. We have engineered site-specific pauses of RNA polymerase II and shown that they alter splicing efficiency and alternative splicing, by unknown mechanism(s) that we will dissect. We will also explore in detail the role of splicing noise (stochastic variations in splicing output over time) on the ability of splicing to control stable homeostatic expression settings (as it does in many RNA binding protein genes) as well as to control a bistable switch (as it does in the Drosophila Sex lethal gene). These experiments will define the operational principles of simple splicing regulatory circuits. The third area of investigation is focused on the process of intron gain and its roles in eukaryotic gene creation and gene diversification. Our recent discovery that the spliceosome can convert the lariat intron to a true intron circle after splicing indicates that it can carry out reverse splicing reactions in vivo, raising questions about whether and how it might promote formation of new introns. We propose to test biochemical steps predicted to be necessary for spliceosome-mediated intron gain, and have already set up experiments to document intron gain in vivo. Given the fundamental conservation of the splicing machinery, this work promises to translate directly into new understanding of the mechanisms of gene regulation in eukaryotes, including humans. Defects in splicing are frequently recognized as contributors to disease, and interventions that address splicing defects are increasingly successful pathways to treatment.

项目总结