Recent technological advances have revealed the existence of thousands of microproteins (<100 amino acids) missing from genome annotations, but little is known about their function. The physiological relevance of this ‘biological dark matter’ is one of the biggest outstanding mysteries in modern biology. Compared to canonical proteins, microproteins evolve at a strikingly fast pace. They frequently appear de novo and rapidly disappear through disabling mutations, overwhelmingly exhibiting evolutionarily novel sequences found in only one species or lineage. The lack of evolutionary conservation renders homology-based approaches for functional prediction powerless and raises the concern that many microproteins might not be functional. The selective pressures driving the rapid evolution of microproteins are largely unknown. In mammals, one of the most well- characterized determinants of protein evolution is the immune system. Novel sequences are positively selected when they mediate functional innovations that enable cells to mount effective innate immune responses to fast evolving pathogens. Novel sequences are negatively selected when are recognized as foreign by the adaptive immune system. We reasoned that the tremendous strength of these immune selective pressures is likely to drive the rapid evolution of microproteins. We therefore propose that the immune system is a critical determinant of microprotein function and evolution. We hypothesize that novel microproteins can only evolve to perform cell-intrinsic functions if they are recognized as ‘self’, and thereby tolerated, by the adaptive immune system. The lineage-specific sequences of these novel but tolerated microproteins would provide a vital arsenal against rapidly evolving pathogens. Conversely, novel microproteins that are recognized as ‘non-self’ would induce auto-immune responses and rapidly disappear over evolutionary time. In this project, we will test the above hypotheses at a proteome-wide level using well established cellular and animal model systems. We will combine: 1) Integrative ribosome profiling to generate a reference microprotein expression atlas in mice; 2) T cell antigen discovery approaches and mouse models of autoimmunity to assess the immunogenic potential of microproteins; 3) Genome-scale gain- and loss-of- function genetic screens to determine cell-intrinsic, innate immune roles of microproteins; and 4) Computational evolutionary genomics to reconstruct the evolutionary history, and estimate the strength of selective pressures acting on microproteins. Our proposed work is centered on mouse models and murine cells due to availability of specific genetic knock-out models and large quantities of matched tissues, plethora of published transcriptome and translatome datasets, and ability to perform experiments in a controlled, homogeneous setting with defined immune genetics. Together, these approaches will illuminate the evolutionary and immunological principles that govern the large, but uncharacterized universe of microproteins and identify microproteins with innate immune function or autoimmune potential.

PROJECT NARRATIVE A major function of the immune system is to discriminate between self and non-self, but occasionally immune cells mistake self-molecules for non-self and attack the body’s own healthy cells, triggering auto-immune diseases. In most cases, the identity of the self-molecules that trigger auto-immunity is unknown. This project will explore the auto-immune potential of an entirely uncharted pool of self-molecules, called ‘microproteins’, with the long-term goals to unlock novel therapeutic strategies for autoimmune diseases and understand the forces that govern microprotein biology.