# Unraveling microprotein biology with an evolutionary-immunological framework

> **NIH NIH R01** · UNIVERSITY OF PITTSBURGH AT PITTSBURGH · 2024 · $1,485,481

## Abstract

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 g...

## Key facts

- **NIH application ID:** 10925317
- **Project number:** 5R01AT012826-02
- **Recipient organization:** UNIVERSITY OF PITTSBURGH AT PITTSBURGH
- **Principal Investigator:** Anne-Ruxandra Carvunis
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $1,485,481
- **Award type:** 5
- **Project period:** 2023-09-08 → 2028-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10925317

## Citation

> US National Institutes of Health, RePORTER application 10925317, Unraveling microprotein biology with an evolutionary-immunological framework (5R01AT012826-02). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10925317. Licensed CC0.

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