Function of novel proteins encoded in non-canonical open reading frames

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Proteins are key players in basically all cellular processes, where they exert a plethora of functions (e.g. catalysis of chemical reactions, transport of molecules). Understanding these functions informs us on the way our cells, organs and bodies work. For decades, it was thought that only one protein was encoded per eukaryotic gene. However, many studies in the past few years have challenged that view, and it is now evident that many genes actually encode multiple proteins. These previously unknown and ignored novel proteins, which are different from well-known protein isoforms, are called alternative proteins and are expressed in our cells in parallel to those already identified (called reference proteins), greatly expanding the protein repertoire. Despite the characterization of a small number of alternative proteins which revealed important biological functions, only a few research groups worldwide consider their existence in their work. Given their recent discovery, alternative proteins remain largely understudied, and are absent from commonly used functional genomics tools. The long-term objective of my research program is to elucidate the molecular, cellular, and organismal functions of alternative proteins, by studying specific examples (candidate approach) and using functional genomics (genome-wide approach). In the present Discovery proposal, we will focus on the following short-term objectives: 1. Determine the molecular, cellular and organismal function of SLC35A4, a gene encoding two proteins, including a strikingly abundant but uncharacterized alternative protein with possible ties to myelin, a nervous system component needed for transit of information. This will be achieved using cultured cells (including human induced pluripotent stem cells) and mouse models, coupled to biochemical, cell biology and multi-omics methods. 2. Create functional genomics tools for genome-wide CRISPR/Cas9 screening to discover the function of alternative proteins at a large scale, and use multi-omics methods to mechanistically characterize functional alternative proteins within the broader cellular landscape. Our findings on the function of our candidate gene (Objective 1) will provide the first deep characterization of this abundant alternative proteins in vertebrates. The cellular and mouse models created will be of interest for other groups to study this dual coding gene (for ex. in the myelination field). Objective 1 will exemplify the necessity of studying both alternative and reference proteins to fully understand the functions of our genes. In addition Objective 2 will significantly accelerate the researchers' ability to study the function of alternative proteins at a large scale. These novel and open functional genomics tools that integrate alternative proteins will significantly help advancing our knowledge of the contribution of alternative proteins to the function of our genes, and will place Canada at the forefront of this new field.