# Functions of mRNA Pseudouridylation

> **NIH NIH R01** · YALE UNIVERSITY · 2023 · $345,050

## Abstract

PROJECT SUMMARY
Multiple pseudouridine synthases (PUS) are implicated in human disease, but the mechanisms
that connect loss of PUS activity to mitochondrial myopathy, digestive disorders, intellectual
disability, resistance to viral infection, dyskeratosis congenita, and diverse cancers remain
largely unknown. There are several critical gaps in our current knowledge of the functions of
PUS proteins. Although the basic biochemical activity of PUS proteins in catalyzing the
isomerization of uridine to pseudouridine is well understood, the specific RNA targets of most
human PUS proteins are unknown or incompletely known. Our long-term goals include
identifying the targets of all PUS proteins and determining the molecular consequences of
modification of specific RNAs with pseudouridine in disease-relevant cellular contexts. This will
be critical for understanding the etiology of diseases caused by PUS deficiency and may reveal
new therapeutic targets for treatment.
Our recent work (Martinez et al. Mol Cell 2022) established that pseudouridine (Ψ) is deposited
co-transcriptionally in nascent human pre-mRNA at thousands of sites where it is poised to
affect every step of mRNA metabolism from birth to death. This renewal application will
rigorously investigate the biochemical basis for mRNA regulation by pseudouridylation. The
biological effects of Ψ must originate in chemical differences between U and Ψ, which directly
affect RNA backbone conformation, the stability of base pairs, and the binding of proteins. Our
preliminary data establish that hundreds of pseudouridines are present within the experimentally
determined binding sites of regulatory RNA-binding proteins (RBPs) and micro RNAs (miRNAs)
in human HepG2 cells. Widespread occurrence of Ψ in RBP and miRNA binding sites—together
with biochemical studies of artificially pseudouridylated RNAs that demonstrate the capacity of
Ψ to determine whether, and to what extent, an mRNA will be bound—establish a strong
premise for the proposed work. We will (1) Determine impact of mRNA Ψ on functional
interactions with regulatory RNA-binding proteins, (2) Identify the roles of pseudouridylation in
miRNA-mediated gene regulation, and (3) Define the contributions of regulated RNA
pseudouridylation to the control of gene expression.
Together, the proposed work will elucidate the molecular consequences of mRNA
pseudouridylation in human cells and define functionally relevant mRNA targets of human
pseudouridine synthases. This will fill a critical knowledge gap for understanding how RNA
modifying enzymes that are essential for human health control gene expression.

## Key facts

- **NIH application ID:** 10659705
- **Project number:** 2R01GM101316-09
- **Recipient organization:** YALE UNIVERSITY
- **Principal Investigator:** Wendy Victoria Gilbert
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $345,050
- **Award type:** 2
- **Project period:** 2014-09-01 → 2027-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10659705

## Citation

> US National Institutes of Health, RePORTER application 10659705, Functions of mRNA Pseudouridylation (2R01GM101316-09). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10659705. Licensed CC0.

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