# Elucidating premature translation termination in Cystic Fibrosis

> **NIH NIH F31** · UNIV OF MASSACHUSETTS MED SCH WORCESTER · 2020 · $32,762

## Abstract

PROJECT SUMMARY
A leading cause of Cystic Fibrosis (CF) is premature termination codons (PTCs) in the cystic fibrosis
transmembrane conductance regulator (CFTR) gene. Suppression of translation termination at PTCs—i.e. PTC
readthrough—to restore full-length CFTR protein may be a treatment strategy. Yet, current PTC readthrough
drug candidates for CF are toxic (e.g. aminoglycosides) or ineffective (e.g. ataluren). Efficacy of PTC readthrough
depends on efficiency of translation termination at the PTC. Thus, manipulating the molecular mechanisms of
CFTR PTC termination to lower efficiency may improve PTC readthrough efficacy. However, strategies for such
manipulations are limited in the absence of a detailed understanding of translation termination on CFTR PTCs.
In the current model for normal termination, eukaryotic Release Factors 1 and 3 form a complex (eRF1•eRF3)
that releases a newly synthesized protein from the ribosome. eRF1 recognizes a tetra-nucleotide stop codon at
the end of an open reading frame, and catalyzes peptidyl-tRNA hydrolysis. Poly-A binding protein (PABP), which
binds at 3′ ends of mRNA, recruits eRF3 and enhances termination efficiency. However, it remains unclear how
PABP, eRF1, eRF3, and the tetra-nucleotide stop codon recognize the PTC to produce truncated CFTR protein.
The goal of this proposal is to determine the biochemical and structural mechanism of translation termination.
With guidance from Dr. Andrei Korostelev (expert in biochemical and structural mechanisms of translation), Dr.
Allan Jacobson (expert in premature translation termination and PTC read-through), Dr. Phillip Zamore (RNA
biochemist), Dr. Chen Xu (cryo-EM instrumentalist), and Dr. Nikolaus Grigorieff (expert in cryo-EM method
development), release assays will be optimized to study the efficiency of translation termination mediated by
eukaryotic release factors, and ensemble time-resolved (ENTIRE) cryo-EM will be used to capture structural
intermediates of enzymatic reactions. Aim 1 will use defined mammalian translation systems to measure the
individual effects of stop codon context, eRF1•eRF3, and PABP on the termination efficiencies (kcat/KM) of CFTR
PTCs and the true stop codon. Aim 2 will visualize how the ribosome terminates on CFTR PTC G542X in its
natural sequence context using ENTIRE cryo-EM. Collecting data at multiple time points will identify
conformational changes and interactions between mRNA sequence, eRF1•eRF3, and PABP during termination.
To reveal the termination mechanism on CFTR PTCs, structures and their progression intermediates will be
compared with those recorded on the true CFTR stop codon. If successful, this study will reveal key molecular
determinants of CFTR PTC termination, and may inform strategies to induce PTC readthrough for CF treatment.

## Key facts

- **NIH application ID:** 9992906
- **Project number:** 1F31HL152650-01
- **Recipient organization:** UNIV OF MASSACHUSETTS MED SCH WORCESTER
- **Principal Investigator:** Christine Elizabeth Carbone
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $32,762
- **Award type:** 1
- **Project period:** 2020-05-01 → 2023-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9992906

## Citation

> US National Institutes of Health, RePORTER application 9992906, Elucidating premature translation termination in Cystic Fibrosis (1F31HL152650-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9992906. Licensed CC0.

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