Uncovering the Function and Protein Subcellular Localization of Bicistronic Loci in Auxenochlorella protothecoides - Project Summary Polycistronic expression is the production of two or more polypeptides from one mRNA. It has historically been assumed that eukaryotic mRNAs are monocistronic. However, applications of advancements in sequencing technology have revealed a growing number of nucleus-encoded polycistronic genes previously undetected in eukaryotic genomes, including hundreds of polycistronic transcripts in the green algal lineage, discovered by the sponsor. The polycistronic arrangement of these genes is conserved over half a billion years of evolution in the green algal radiation, and the proteins encoded by these loci are conserved throughout eukaryotes, including humans. In previous work, the applicant addressed the question of the mechanism of translation of two proteins from a single RNA, and concluded, based on bioinformatic analysis and experimental tests, that leaky ribosome scanning was the most likely mechanism. Now, the applicant will address the actual function of the proteins encoded by two polycistronic loci. In some cases, where the function of the locus is known, the two proteins function together in the same pathway. Nevertheless, in many cases the proteins’ function is unknown, which offers the applicant an opportunity for discovery. In Aim 1, the applicant will use facile gene replacement methodology, developed for the green alga Auxenochlorella protothecoides in the sponsor’s laboratory, for reverse genetic analysis of two polycistronic loci. For each locus, strains carrying individual loss of function ORF1 and ORF2 as well as dual loss of function ORF1+ORF2 strains will be created. The mutants will be compared to the wild-type for phenotyping, which will involve physiological as well as biochemical assays focused on bioenergetic and metabolic parameters related to photosynthesis and respiration, located respectively in the chloroplast and mitochondria. Besides deducing the function(s) of these conserved proteins the work may also reveal why the proteins remain polycistronic. In parallel, in Aim 2, the applicant will develop fluorescent protein reporters to mark individual organellar compartments in A. protothecoides, based on well-characterized proteins and reporter fusions in classic model organisms. These can be used as positive controls for localization of the ORFs of interest from Aim 1, which can reinforce the deduced function(s). The reporters will also serve as a community resource for discovery projects or for synthetic biology designs. The sponsor and institution offer the resources and expertise for executing the proposed research, have programs for and are committed to mentoring the applicant. The training program includes development of soft skills and adherence to professional standards. The applicant will receive an intellectual foundation, scientific independence, and a diverse set of skills, all of which will prepare him to be an exceptional independent investigator.
