# Self-cleaving peptides: Mechanisms and Use in Diverse Eukaryotic Species

> **NIH NIH R21** · UNIVERSITY OF COLORADO DENVER · 2023 · $191,448

## Abstract

SUMMARY
Giardia lamblia is a single-cell eukaryote that infects hundreds of millions of people every year. Because Giardia
has many molecular pathways that are simplified compared to other eukaryotes, it has potential as a
nontraditional model system for studying the diversity and evolution of key biological processes.
 We recently serendipitously discovered that the 2A ‘self-cleaving’ peptide sequences work very poorly in
Giardia, surprising because 2A peptides are thought to work universally in eukaryotes. Found in picornaviruses
like foot-and-mouth disease virus and poliovirus, 2A peptides are an essential part of the viral life cycle because
they enable two polypeptides to be produced from one open reading frame. Although often referred to as ‘self-
cleaving,’ 2A peptides operate by causing the ribosome to skip a peptide bond. The mechanism of this is
unknown but must involve specific interactions between the 2A nascent peptide chain and the exit tunnel of the
ribosome. Thus, our discovery that 2A peptides work poorly in Giardia points at fundamental differences in its
ribosomes compared to other eukaryotes and can be exploited to understand the mechanism of 2A action.
 Examination of our recently solved structure of the Giardia 80S ribosome reveals a compelling difference
in the structure of ribosome protein uL4 in the exit channel: Giardia lacks a specific loop in uL4. We hypothesize
that this loop is important for the peptide bond-skipping mechanism of 2A peptides, and its absences can partially
explain why 2A peptides operate poorly in Giardia. Here, we will test this hypothesis and in so doing (1) define
the mechanism by which 2A peptides induce bond skipping and (2) determine why it fails in Giardia. We will
combine genetic, biochemical, and structural approaches in two aims. In the first aim, we will determine the
extent to which 2A sequence variants can function in Giardia, with the goal of finding novel efficient and functional
sequences that will serve as powerful tools for Giardia researchers. In the second aim, we will directly test the
functional role of the uL4 loop and solve the structure of a T2A-ribosome complex by cryo-EM with the goal of
describing the mechanism of peptide bond skipping in eukaryotes. Overall, this work will provide critical
knowledge about the function of the Giardia ribosome, the fundamental workings of the eukaryotic translational
machinery, and the mechanism of 2A peptide function. Our discoveries will facilitate the development of Giardia
as a model organism and help lay the foundation for new anti-viral therapeutics that block 2A peptide activity.

## Key facts

- **NIH application ID:** 10678481
- **Project number:** 1R21AI167423-01A1
- **Recipient organization:** UNIVERSITY OF COLORADO DENVER
- **Principal Investigator:** Olivia Selfridge Rissland
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $191,448
- **Award type:** 1
- **Project period:** 2023-09-01 → 2025-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10678481

## Citation

> US National Institutes of Health, RePORTER application 10678481, Self-cleaving peptides: Mechanisms and Use in Diverse Eukaryotic Species (1R21AI167423-01A1). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10678481. Licensed CC0.

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