# Functions of mRNA Pseudouridylation

> **NIH NIH R01** · YALE UNIVERSITY · 2021 · $335,000

## 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 previous studies revealed that pseudouridine is a prevalent modification of nascent
pre-messenger RNA. These results, together with quantitative in vitro studies showing that
pseudouridine can affect both RNA-protein and RNA-RNA interactions, lead to our central
hypothesis that pre-mRNA pseudouridylation controls human gene expression at the level of
pre-mRNA splicing. In support of this hypothesis, we have demonstrated that loss of one pre-
mRNA modifying pseudouridine synthase, PUS1, causes widespread changes to pre-mRNA
splicing in human cells, with more than 3,000 PUS1-sensitive alternative splicing events
identified. Our Specific Aims are to (1) Define the splicing-relevant pre-mRNA targets of the
predominant pre-mRNA pseudouridylating enzymes: PUS1, PUS7, and RPUSD4; and (2)
Elucidate the molecular mechanisms of pseudouridine-sensitive splicing.
 The proposed work will provide key insight into the molecular functions of
pseudouridines in pre-mRNAs and may reveal novel modes of eukaryotic gene regulation. By
establishing pre-mRNAs as a broad new class of substrates for PUS enzymes, our work
implicates defective splicing as a plausible but understudied mechanism connecting loss of PUS
activity to numerous human diseases.

## Key facts

- **NIH application ID:** 10206158
- **Project number:** 5R01GM101316-07
- **Recipient organization:** YALE UNIVERSITY
- **Principal Investigator:** Wendy Victoria Gilbert
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $335,000
- **Award type:** 5
- **Project period:** 2014-09-01 → 2023-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10206158

## Citation

> US National Institutes of Health, RePORTER application 10206158, Functions of mRNA Pseudouridylation (5R01GM101316-07). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10206158. Licensed CC0.

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