# IRES-mediated translation: mechanisms and applications in mRNA therapeutics

> **NIH NIH R35** · UNIV OF MASSACHUSETTS MED SCH WORCESTER · 2024 · $418,750

## Abstract

SUMMARY
Protein synthesis is primarily regulated at the initiation stage, allowing rapid and reversible control of gene
expression. Whereas cellular mRNAs require a 5′ cap to initiate translation, many viral mRNAs require an internal
ribosome entry site (IRES). IRESs fold into three-dimensional structures and initiate translation through non-
canonical interactions with cellular translation initiation machinery. Despite decades of research, many important
questions remain unanswered about how IRESs initiate translation, particularly for the structurally complex Type
I and Type II IRESs. Using biochemical studies, we have observed previously unknown IRES RNA structural
changes during translation initiation. We have also leveraged powerful cryo-EM techniques to obtain high-
resolution Type II IRES structures at the final stage of initiation. In the first theme of this proposal, we will build
upon these results and integrate biochemical, chemical, molecular, and structural techniques to determine how
IRES structural dynamics regulate translation initiation of Type I and Type II IRESs. Our long-term goal is to
combine these mechanistic insights with high-throughput RNA structural probing and translation complex
profiling to understand how natural IRES mutations affect structural dynamics, translation efficiency, and
virulence.
 Type I and Type II IRESs are crucial for circular mRNA (circRNA) technology, which has gained interest
as a new mRNA therapeutic platform. Current circRNA design will benefit from incorporating modified
nucleosides, such as pseudouridine (ψ) or N1-methylpseudouridine (m1ψ), that are critical for reducing the
immunogenicity of linear mRNAs. However, ψ/m1ψ modification inactivates Group I introns and Type I/II IRESs—
key RNA elements for circularizing and translating circRNAs, respectively. This result is paradoxical given that
uridine, ψ, and m1ψ are virtually interchangeable in transcription and translation due to their structural similarities.
Our overall hypothesis is that while these modifications maintain and stabilize the Watson-Crick base pairing,
they disrupt certain non-canonical base pairing or tertiary interactions critical for folding large non-coding RNAs.
The goal of the second theme of our research is to solve this paradox and determine how ψ and m1ψ affect RNA
structures and translation. Using the Group I intron and Type II IRES as our model molecular systems, we will
use RNA structural probing, biochemical, and molecular techniques to elucidate how these modified nucleotides
affect RNA tertiary interactions and folding beyond canonical Watson-Crick base pairing. Our results will provide
fundamental knowledge of how base modifications affect RNA folding and function, informing the development
of potent mRNA therapeutics.

## Key facts

- **NIH application ID:** 10941603
- **Project number:** 1R35GM155169-01
- **Recipient organization:** UNIV OF MASSACHUSETTS MED SCH WORCESTER
- **Principal Investigator:** Li Li
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $418,750
- **Award type:** 1
- **Project period:** 2024-07-05 → 2029-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10941603

## Citation

> US National Institutes of Health, RePORTER application 10941603, IRES-mediated translation: mechanisms and applications in mRNA therapeutics (1R35GM155169-01). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10941603. Licensed CC0.

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