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

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。这个过程的一个重要步骤是前mrna剪接，在这个过程中，中断序列从新生转录物中移除。通过替代mRNA区域的包含，pre-mRNA剪接极大地拓宽了特定基因编码的蛋白质范围，并提供了额外的调控层，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，并且对后生动物和真菌有很强的分类偏见。发表的结果：人类和酵母剪接系统已被证明是非常复杂的详细机制研究。我假设基因组较小的极端微生物可能有一套更简单的拼接机制。我和我的学生们在单细胞红藻C. merolae中发现了一种简单得多的剪接体，它只由大约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，这种剪接粒子通常执行底物识别的第一步。目的：我的长期目标是确定每个剪接成分在C. merolae中的作用，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和研究它们的功能来利用我们简化的拼接系统的力量。科学方法：我们将使用一系列新的C. merolae特异性试剂来分离和表征剪接复合物，并研究剪接决策在不同条件下如何改变C. merolae的适合度。目的1：纯化和表征内源性复合物，并测试它们在结合底物和催化剪接中的功能。目的2：确定C. merolae中的剪接是否以及如何被调节。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，并测量对生长和应激反应的影响。可行性：补充了多年来在酵母中剪接生物化学的研究，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，我们已经开发了一些试剂（抗体，转化方法，morpholino oligos），这些试剂将用于拟议的工作。新颖性和重要性：我的实验室正在用C. merolae开发多年来研究剪接的第一个新模型系统，这是一个独特的系统，因为它的简单性和进化意义。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.