Studies of mRNA Expression and Degradation

Citation

Gálvez Merchán, Ángel (2023) Studies of mRNA Expression and Degradation. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/esxk-ch24. https://resolver.caltech.edu/CaltechTHESIS:06042023-195408313

Abstract

Part 1: Protein degradation coupled to Nonsense-mediated mRNA decay

Translation of mRNAs containing premature termination codons (PTCs) results in truncated protein products with deleterious effects. Nonsense-mediated decay (NMD) is a surveillance pathway responsible for detecting PTC containing transcripts. While the molecular mechanisms governing mRNA degradation have been extensively studied, the fate of the nascent protein product remains largely uncharacterized. In part 1 of this thesis, we use a fluorescent reporter system in mammalian cells to reveal a selective degradation pathway specifically targeting the protein product of an NMD mRNA. We show that this process is post-translational, and dependent on the ubiquitin proteasome system. To systematically uncover factors involved in NMD-linked protein quality control, we conducted genome-wide flow cytometry-based screens. Our screens recovered known NMD factors, but suggested protein degradation did not depend on the canonical ribosome-quality control (RQC) pathway. A subsequent arrayed screen demonstrated that protein and mRNA branches of NMD rely on a shared recognition event. Our results establish the existence of a targeted pathway for nascent protein degradation from PTC containing mRNAs, and provides a reference for the field to identify and characterize required factors.

Part 2: The Commons Cell Atlas

Current cell atlas projects aim to curate representative datasets, cell-types, and marker genes for tissues across an organism. Despite their ubiquity, atlas projects rely on duplicated and manual effort to curate marker genes and annotate cell-types. Importantly, the lack of data-compatible tools and a fixed representation of the atlas make their reanalysis near-impossible. To overcome these challenges, we present a collection of data, algorithms, and tools to automate cataloging and analyzing cell-types across all tissues in an organism. We leveraged this work to build a Human Commons Cell Atlas comprising 2.9 million cells across 27 tissues that can be easily updated and that is structured to facilitate custom analyses. To showcase the flexibility of the atlas, we demonstrate that it can be used for isoform analyses. In particular, we study cell-type specificity of isoforms of OAS1, which has recently been shown to offer SARS-CoV-2 protection in certain individuals that display higher expression of the p46 isoform. Using our Commons Cell Atlas, we localize the OAS1 p44b isoform to the testis, and find that it is specific to germ line cells. By virtue of enabling customized analyses via a modular and dynamic atlas structure, the Commons Cell Atlas should be useful for exploratory analyses that are intractable within the rigid framework of current gene-centric static atlases.

Thesis Files