Mechanism and regulation of splicing in the dramatically reduced spliceosome of C. merolae

C. merolae 剪接体显着减少的剪接机制和调控

• 批准号: RGPIN-2017-04783
• 负责人: Rader, Stephen
• 金额: $ 2.91万
• 依托单位: University of Northern British Columbia
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=711217
• 关键词: Mechanism; regulation; splicing; dramatically; reduced

From the creation of an mRNA transcript to the degradation of the protein it encodes, there are a multitude of regulatory steps in eukaryotic gene expression. One essential step in this process is pre-mRNA splicing, in which interrupting sequences are removed from nascent transcripts. Through alternative inclusion of mRNA regions, pre-mRNA splicing dramatically broadens the range of proteins encoded by a particular gene, and provides additional regulatory layers that allow a cell to respond to environmental conditions, developmental signals, and cell cycle progression. Our understanding of this process, however, comes from a very limited range of model organisms with a strong taxonomic bias towards metazoans and fungi. Published results: Human and yeast splicing systems have proven intractably complex for detailed mechanistic studies. I hypothesized that an extremophile with a small genome might have a simpler set of splicing machinery. With my students, I have identified in the unicellular red alga C. merolae a dramatically simpler spliceosome consisting of only ~40 core proteins and four snRNAs. Strikingly, it is the first organism demonstrated to lack the U1 snRNP, the splicing particle that normally carries out the first step of substrate recognition. Objectives: My long-term objectives are to determine the role of each splicing component in C. merolae, as well as to understand the regulation, biological consequences, and evolution of splicing. My short-term objectives are to exploit the power of our simplified splicing system by determining structures of its components and investigating their function. Scientific approach: We will use a battery of new C. merolae-specific reagents to isolate and characterize splicing complexes and to study how splicing decisions alter the fitness of C. merolae under different conditions. Aim #1: purify and characterize endogenous complexes and test their function in binding substrates and catalyzing splicing. Aim #2: determine whether and how splicing is regulated in C. merolae. Aim #3: investigate the biological relevance of splicing by using CRISPR-Cas9 to delete introns and measure the effect on growth and stress responses. Feasibility: Complementing years of splicing biochemistry in yeast, we have successfully isolated and characterized splicing complexes from C. merolae in published experiments. In addition, we have developed a number of reagents (antibodies, transformation methods, morpholino oligos) that will be required for the proposed work. Novelty and significance: With C. merolae, my lab is developing the first new model system for studying splicing in years, and a unique system for its simplicity and evolutionary implications. These discoveries will have broadly felt impacts on technologies based on gene expression, a wide range of human and animal diseases, and fundamental questions of how splicing has evolved.

从 mRNA 转录物的产生到其编码的蛋白质的降解，真核基因表达中有许多调控步骤。这一过程中的一个重要步骤是前 mRNA 剪接，其中中断序列从新生转录本中被去除。通过选择性地包含 mRNA 区域，前 mRNA 剪接极大地拓宽了特定基因编码的蛋白质范围，并提供了额外的调节层，使细胞能够对环境条件、发育信号和细胞周期进程做出反应。然而，我们对这一过程的理解来自于非常有限的模式生物，这些模式生物对后生动物和真菌有强烈的分类学偏见。 已发表的结果：人类和酵母剪接系统已被证明对于详细的机制研究而言极其复杂。我假设基因组较小的极端微生物可能拥有一套更简单的剪接机制。我和我的学生一起在单细胞红藻 C. merolae 中发现了一个极其简单的剪接体，仅由约 40 个核心蛋白和 4 个 snRNA 组成。引人注目的是，它是第一个被证明缺乏 U1 snRNP 的生物体，U1 snRNP 是通常执行底物识别第一步的剪接颗粒。 目标：我的长期目标是确定 C. merolae 中每个剪接成分的作用，以及了解剪接的调节、生物学后果和进化。我的短期目标是通过确定其组件的结构并研究其功能来利用我们简化拼接系统的强大功能。 科学方法：我们将使用一系列新型 C. merolae 特异性试剂来分离和表征剪接复合物，并研究剪接决策如何改变 C. merolae 在不同条件下的适应性。 目标#1：纯化和表征内源复合物并测试其在结合底物和催化剪接中的功能。 目标#2：确定剪接是否以及如何在 C. merolae 中受到调节。 目标#3：通过使用 CRISPR-Cas9 删除内含子来研究剪接的生物学相关性，并测量对生长和应激反应的影响。 可行性：作为对酵母中多年剪接生物化学研究的补充，我们在已发表的实验中成功地从 C. merolae 中分离和表征了剪接复合物。此外，我们还开发了拟议工作所需的许多试剂（抗体、转化方法、吗啉寡核苷酸）。 新颖性和意义：通过 C. merolae，我的实验室正在开发多年来第一个用于研究剪接的新模型系统，并且是一个因其简单性和进化意义而独特的系统。这些发现将对基于基因表达的技术、广泛的人类和动物疾病以及剪接如何进化的基本问题产生广泛的影响。