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

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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.