Regulation and function of the hidden proteome in mammalian cells
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ly-jimmyly-phd-biology-2025-thesis .pdf
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Author(s)
Ly, Jimmy
Advisor(s)
Cheeseman, Iain
Date Issued
February 2026
Publisher
Massachusetts Institute of Technology
Abstract
The human body is composed of trillions of cells and exhibits remarkable biological complexity—far exceeding that of single-celled organisms like yeast or bacteria. Yet our genome contains only about 20,000 protein-coding genes, just a few times more than that of many bacteria. So where does this complexity arise? The answer lies not in the number of our genes, but in how we use them. Rather than producing a single protein from each gene, human cells employ multiple mechanisms to generate alternative, “hidden” protein variants—previously overlooked products of our own genome. Several molecular mechanisms enable this diversity. One well-established mechanism is alternative splicing, which produces multiple mRNA isoforms from a single gene, leading to distinct protein products. Another, more emerging mechanism involves alternative translation initiation, where the ribosome selects among multiple potential start codons on a single mRNA. Although translation canonically begins at an “AUG” codon, the ribosome can initiate at multiple start codons in a single mRNA—including non-AUG start codons— producing protein isoforms that share the same core sequence but differ at their N-termini. These alternative N-terminal isoforms—part of the “hidden proteome”—represent a vast and largely unexplored layer of biological complexity. Here, I combined high-throughput, molecular, and cell biological approaches to uncover the regulation, function, and physiological relevance of the hidden proteome. First, I characterized a specific alternative splicing event in a key cell division factor during meiosis and demonstrated its importance for fertility. I then discovered a global, cell cycle–dependent switch in alternative translation initiation. I show that this translational remodeling is mediated by the sequestration of the translation stringency factor eIF1 in the nucleus during interphase and its release into the cytoplasm during mitosis. Building on these findings, I demonstrated that alternative translation is a powerful and widespread mechanism for generating protein isoforms with distinct subcellular localizations. I further leveraged these insights to highlight the significance of alternative start codon selection in interpreting rare disease variants, introducing the concept of isoform-selective alleles. Finally, I expanded my analyses beyond alternative isoforms to investigate the roles of previously uncharacterized genes, identifying two novel factors essential for ribosome biogenesis and contributing to the systematic functional annotation of the genome. Together, these studies reveal fundamental principles governing the regulation and function of the “hidden proteome,” offering new perspectives on gene expression, cellular complexity, development, and human disease.
MIT Department
Massachusetts Institute of Technology. Department of Biology
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