Redefining Criteria for RNA-Binding Activity Through Signal-to-Noise (S:N)-Based Analysis of RNA-Bound Proteomes

dc.contributor.advisor

Nicchitta, Christopher V

dc.contributor.author

Kristofich, JohnCarlo Louis

dc.date.accessioned

2024-03-07T18:39:24Z

dc.date.issued

2023

dc.department

Cell Biology

dc.description.abstract

RNA-binding proteins (RBPs) play an important role in post-transcriptional gene regulation by binding mRNAs to regulate splicing, stability, nuclear export, and target them for translation at specific subcellular compartments. While the role of RBPs in localization of mRNA to different subcellular compartments has been expanding in recent years, little has been done to understand their role in targeting mRNA for translation at the ER. The recent introduction of UV-based RNA-centric methods for proteome-wide identification of RNA-interactors has revealed numerous candidate RBPs on the ER. However, validation for most of these candidate integral ER RBPs is lacking, and the biological function of their RNA-binding activity is unknown. To address this, we introduce signal-to-noise (S:N)-based analytical approaches for direct validation of RNA-binding and reveal previously unrecognized sources of experimental noise inherit to UV-based RNA-centric methods which confound meaningful interpretation and introduce false positives. To overcomes these issues, we devise a novel biochemical method termed LEAP-RBP (Liquid-Emulsion-Assisted-Purification of RNA-Bound Protein) for the selective isolation of UV-crosslinked RNA-protein adducts and introduce new signal-to-noise (S:N)-based metrics for distinguishing relevant vs non-specific RNA-interactors. Using this approach, we identify integral ER RBPs with potential RNA-regulatory roles and provide a solid foundation for the proper identification and characterization of RNA-protein interactomes.

dc.identifier.uri

https://hdl.handle.net/10161/30307

dc.rights.uri

https://creativecommons.org/licenses/by-nc-nd/4.0/

dc.subject

Biology

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Endoplasmic reticulum

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RBP

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RNA

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RNA binding protein

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UV-crosslinking

dc.title

Redefining Criteria for RNA-Binding Activity Through Signal-to-Noise (S:N)-Based Analysis of RNA-Bound Proteomes

dc.type

Dissertation

duke.embargo.months

23

duke.embargo.release

2026-02-07T18:39:24Z

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