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

蛋白质在几乎所有的细胞过程中都是关键角色，它们发挥着过多的功能（例如，化学反应的催化，分子的运输）。了解这些功能可以让我们了解细胞、器官和身体的工作方式。几十年来，人们一直认为每个真核基因只编码一种蛋白质。然而，过去几年的许多研究挑战了这一观点，现在很明显，许多基因实际上编码多种蛋白质。这些以前未知和被忽视的新蛋白质，不同于已知的蛋白质同种异构体，被称为替代蛋白，并在我们的细胞中与已经确定的蛋白质（称为参考蛋白）平行表达，极大地扩展了蛋白质库。尽管少数替代蛋白的特性揭示了重要的生物学功能，但全世界只有少数研究小组在他们的工作中考虑到它们的存在。鉴于他们最近的发现，替代蛋白在很大程度上仍未得到充分研究，并且在常用的功能基因组学工具中缺失。我的研究计划的长期目标是通过研究特定的例子（候选方法）和使用功能基因组学（全基因组方法）来阐明替代蛋白质的分子、细胞和有机体功能。在目前的探索计划中，我们将重点关注以下短期目标：确定SLC35A4的分子、细胞和机体功能，SLC35A4是一种编码两种蛋白质的基因，其中包括一种非常丰富但未被表征的替代蛋白质，可能与髓磷脂有关，髓磷脂是一种信息传递所需的神经系统成分。这将通过培养细胞（包括人类诱导多能干细胞）和小鼠模型，结合生化、细胞生物学和多组学方法来实现。2. 创建用于全基因组CRISPR/Cas9筛选的功能基因组学工具，以大规模发现替代蛋白的功能，并使用多组学方法在更广泛的细胞景观中机械地表征功能替代蛋白。我们对候选基因功能的发现（目标1）将首次深入表征脊椎动物中这种丰富的替代蛋白。创建的细胞和小鼠模型将对其他研究小组研究这种双重编码基因（例如在髓鞘形成领域）感兴趣。目的1将举例说明研究替代蛋白和参考蛋白的必要性，以充分了解我们基因的功能。此外，目标2将显著加快研究人员大规模研究替代蛋白功能的能力。这些新颖而开放的功能基因组学工具整合了替代蛋白，将极大地帮助我们了解替代蛋白对基因功能的贡献，并将加拿大置于这一新领域的前沿。

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.