# Genetic nonsense and its consequences

> **NIH NIH R35** · UNIV OF MASSACHUSETTS MED SCH WORCESTER · 2021 · $562,247

## Abstract

Project Summary/Abstract
 Nonsense-mediated mRNA decay (NMD), the destabilization of an otherwise stable mRNA by
premature translation termination, is a conserved quality control pathway that exemplifies the
interdependence of mRNA decay and protein synthesis. NMD has been extensively studied in multiple
eukaryotes, particularly with respect to the nature of its substrates and the structures and interactions of
its central regulators, Upf1, 2, and 3. In spite of these efforts many key mechanistic questions about this
important regulatory pathway remain to be resolved. We have yet to understand the detailed differences
between normal and premature termination or the molecular events by which the Upf proteins selectively
target translating mRNAs containing premature termination codons (PTCs), promote mRNA
destabilization, or enhance the disassembly of a poorly dissociable premature termination complex. In
part, an understanding of these problems required new approaches that take into account the important
roles played by components of the protein synthesis apparatus in implementing NMD. Using the yeast
Saccharomyces cerevisiae as a model system, we have now established such approaches. We
developed a selective ribosome profiling procedure that allows delineation of the specificity and timing of
Upf factor association with translating ribosomes, combined the selective purification of Upf1-associated
ribosomes with cryo-electron microscopy to localize Upf1 to a specific ribosomal domain, combined mass
spectrometry and efficient purification of full-length proteins derived from nonsense codon readthrough to
elucidate details of aberrant translation termination, and identified and characterized novel negative and
positive regulatory elements, including two Upf1-binding sites, in the previously uncharacterized C-
terminal domain of Dcp2, the catalytic component of the mRNA decapping enzyme. In the experiments of
this proposal, we will follow up on these developments, addressing three principal research directions
that seek to: i) define the mechanistic differences between premature and normal translation termination,
ii) elucidate the function of ribosome-associated Upf proteins, and iii) determine the mechanism of
decapping activation by Upf1 and other decapping activators. At the conclusion of these studies we
anticipate being able to formulate an integrated model detailing the molecular events linking premature
translational termination to targeted mRNA decay.

## Key facts

- **NIH application ID:** 10126862
- **Project number:** 5R35GM122468-05
- **Recipient organization:** UNIV OF MASSACHUSETTS MED SCH WORCESTER
- **Principal Investigator:** Allan S Jacobson
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $562,247
- **Award type:** 5
- **Project period:** 2017-04-01 → 2023-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10126862

## Citation

> US National Institutes of Health, RePORTER application 10126862, Genetic nonsense and its consequences (5R35GM122468-05). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10126862. Licensed CC0.

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