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

从信使核糖核酸转录本的产生到其编码的蛋白质的降解，真核基因的表达有许多调控步骤。在这个过程中，一个重要的步骤是前信使核糖核酸的剪接，在这个过程中，中断序列被从新生转录中移除。通过选择性地包含信使核糖核酸区域，前信使核糖核酸剪接极大地扩大了特定基因编码的蛋白质的范围，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展做出反应。然而，我们对这一过程的理解来自于对后生动物和真菌有强烈分类偏见的非常有限的模式生物。 公布的结果：对于详细的机制研究，人类和酵母剪接系统已被证明是复杂的。我推测，一个基因组很小的嗜极者可能有一套更简单的剪接机制。和我的学生们一起，我在单细胞红藻C.merolae中发现了一个简单得多的剪接体，只由大约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是通常进行底物识别的第一步的剪接颗粒。 目的：我的长期目标是确定每个剪接成分在美罗华弧菌中的作用，以及理解剪接的调节、生物学后果和进化。我的短期目标是通过确定组件的结构和研究它们的功能来利用我们简化的剪接系统的力量。 科学方法：我们将使用一系列新的分枝杆菌特异性试剂来分离和表征剪接复合体，并研究剪接决定如何在不同条件下改变分枝杆菌的适合性。 目的#1：纯化和鉴定内源性复合体，并检测其结合底物和催化剪接的功能。 目的#2：确定剪接是否以及如何在子囊藻中被调控。 目的#3：通过使用CRISPR-Cas9删除内含子来研究剪接的生物学相关性，并测量其对生长和应激反应的影响。 可行性：作为多年酵母剪接生物化学研究的补充，我们在已发表的实验中成功地分离和鉴定了酵母菌的剪接复合体。此外，我们还开发了一些试剂(抗体、转化方法、吗啉低聚物)，这将是拟议工作所需的。 新颖性和意义：我的实验室正在开发多年来第一个研究剪接的新模型系统，以及一个因其简单性和进化意义而独一无二的系统。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。

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.