# Role of pseudouridines in pre-mRNA processing

> **NIH NIH R00** · STANFORD UNIVERSITY · 2023 · $248,999

## Abstract

PROJECT SUMMARY
Recent development of transcriptome-wide approaches to detect modified nucleotides has revealed an
expanding repertoire of modified nucleotides in messenger RNA. RNA modifications represent a new layer of
eukaryotic gene regulation. Pseudouridine (Ψ) is a modified nucleotide that is prevalent in human mRNAs and
can be dynamically regulated in different conditions. However, the endogenous functions of Ψ in mRNA are not
currently understood. We have shown that nascent pre-mRNA is pseudouridylated co-transcriptionally at
thousands of locations by multiple Pseudouridine Synthases (PUSs). We found that pre-mRNA Ψs are
enriched in regions important for splicing regulation and overlap splicing factor binding sites. Genetic
manipulation of Ψ synthases leads to widespread alternative splicing and individual Ψ directly affect splicing in
vitro. PUSs have been linked to a wide range of diseases, including cancer. High expression of pre-mRNA Ψ
synthases correlates with decreased survival in hepatocellular carcinoma cells (HCC) and rewiring of gene
expression by alternative splicing is important for HCC pathology. NIH K99/R00 Pathway to Independence
Award seeks to define PUS-dependent alternative splicing networks in HCC cells (Aim 1), identify molecular
mechanisms by which Ψs regulates pre-mRNA splicing (Aim 2) and investigate mechanisms by which Ψ is
deposited in pre-mRNA co-transcriptionally (Aim 3). During the mentored phase of this award Dr. Martinez will
identify PUS-dependent alternative splicing in HepG2 cells by sequencing based methods for Ψ detection and
alternative splicing analysis.
splicing
Dr. Martinez will determine the effect of Ψ on the binding of candidate Ψ-sensitive
factors, whose binding sites are enriched for overlapping Ψ locations, using biochemical approaches.
During the independent phase, Dr. Martinez will define the HCC specific pre-mRNA Ψ landscape and identify
PUS-dependent HCC specific alternative splicing events by profiling Ψ, and corresponding splicing changes in
primary normal and HCC cells. Her elucidation of mechanisms of Ψ-mediated splicing regulation in HCC cells
will uncover potential disease related modes of splicing regulation. During
the
K99
and
R00
portion
of
the
award
Dr.
Martinez
will
determine
how Ψ synthases are recruited to nascent pre-mRNA by investigating
mechanisms of recruitment to chromatin and association with Pol II by a combination of chromatin- and co-
immunoprecipitation experiments and measuring extent of pseudouridylation in relation to transcription
dynamics using a novel sequencing approach. The pathology of disease-linked Ψ synthases may be due to
dysregulation of their pre-mRNA targets. Therefore, defining and characterizing the effect of PUS-dependent
pre-mRNA Ψs in pre-mRNA processing will help explain how these enzymes contribute to disease. The
Pathway to Independence Award will allow Dr. Martinez to gain the necessary expertise (e.g. technology
development and computational ...

## Key facts

- **NIH application ID:** 10558697
- **Project number:** 5R00GM135537-04
- **Recipient organization:** STANFORD UNIVERSITY
- **Principal Investigator:** Nicole Martinez
- **Activity code:** R00 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $248,999
- **Award type:** 5
- **Project period:** 2020-08-01 → 2025-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10558697

## Citation

> US National Institutes of Health, RePORTER application 10558697, Role of pseudouridines in pre-mRNA processing (5R00GM135537-04). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10558697. Licensed CC0.

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