Revealing unknown micropeptide functions using high-throughput screening
File(s)
Author(s)
Walmsley, Emma
Type
Thesis
Abstract
A hidden world of biological regulation exists in the form of micropeptides, which are defined as polypeptides of 100 amino acids or less that are produced from small opening reading frames (smORFs). These micropeptides impact many important biological processes yet remain largely understudied despite the existence of thousands of uncharacterised smORFs. Additionally, synthetic micropeptides show potential as biotechnological tools for research, diagnostics, and therapeutics due to their specific binding capabilities.
However, research in these fields is limited by a lack of high-throughput methods for identifying micropeptide function. In this thesis, I present a novel screening method to determine micropeptide function through identifying micropeptide-protein interactions. My adapted yeast two-hybrid (Y2H) approach, combining low-labour library construction with next-generation sequencing, is fast, cheap, and scalable. It is high throughput and multiplexed, allowing large libraries to be screened simultaneously in a single experiment generating a wealth of data for further analysis.
I successfully applied my Y2H technique to synthetic micropeptides, identifying hundreds of peptides which specifically bind to proteins of interest, thus providing a novel approach for identifying micropeptides for research and drug development. I then applied my technique to 384 putative micropeptides from Drosophila melanogaster, revealing thousands of micropeptide-protein interactions. These interactions are highlighting micropeptides binding to proteins involved in metabolism, gene expression regulation, and development.
The work in this thesis establishes that my innovative Y2H screening method is a promising approach which has the potential to not only advance not only our understanding of the biological processes being regulated by micropeptides, but also offers a crucial way to advance the emerging field of micropeptide research.
However, research in these fields is limited by a lack of high-throughput methods for identifying micropeptide function. In this thesis, I present a novel screening method to determine micropeptide function through identifying micropeptide-protein interactions. My adapted yeast two-hybrid (Y2H) approach, combining low-labour library construction with next-generation sequencing, is fast, cheap, and scalable. It is high throughput and multiplexed, allowing large libraries to be screened simultaneously in a single experiment generating a wealth of data for further analysis.
I successfully applied my Y2H technique to synthetic micropeptides, identifying hundreds of peptides which specifically bind to proteins of interest, thus providing a novel approach for identifying micropeptides for research and drug development. I then applied my technique to 384 putative micropeptides from Drosophila melanogaster, revealing thousands of micropeptide-protein interactions. These interactions are highlighting micropeptides binding to proteins involved in metabolism, gene expression regulation, and development.
The work in this thesis establishes that my innovative Y2H screening method is a promising approach which has the potential to not only advance not only our understanding of the biological processes being regulated by micropeptides, but also offers a crucial way to advance the emerging field of micropeptide research.
Version
Open Access
Date Issued
2024-05-21
Date Awarded
01/10/2024
License URL
Advisor
Southall, Tony
Aughey, Gabriel
Sponsor
The Leverhulme Trust
Grant Number
RPG-2019-393
Publisher Department
Life Sciences
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)