# Mechanisms for establishment and recognition of stop codon context

> **NIH NIH K99** · JOHNS HOPKINS UNIVERSITY · 2020 · $89,616

## Abstract

Project Summary / Abstract
The efficiency of stop codon recognition and the activation of downstream quality control depend on the RNA
sequence, protein and spatial contexts of a particular stop codon. Genetic mutations and defects in messenger
RNA (mRNA) processing often result in premature termination codons (PTCs). Translating these mRNAs
produces truncated proteins that often have deleterious effects for the cell or organism. The effects of such
mutations are buffered by nonsense-mediated mRNA decay (NMD), which selectively degrades mRNAs with
PTCs, as well as hundreds of “normal” mRNA species. Current models fail to explain how most PTCs are
detected, particularly the molecular details that connect the mechanics of translation termination to mRNA decay.
Stop codon readthrough, even at relatively low efficiency, can render a message resistant to NMD. There is also
evidence that underlying mechanisms are shared between stop codon recognition and NMD. The goal of this
proposal is to identify currently unknown factors involved in NMD and stop codon readthrough and to understand
their biochemical roles in these processes, especially as it pertains to modulating the activity of ribosomes.
In Aim 1 I will use CRISPR-Cas9 screening and mass spectrometry approaches to identify candidate protein
factors that differentiate PTCs from normal stop codons and contribute to stop codon readthrough in vivo. The
genes identified in these screens are potential targets for therapeutic intervention in genetic diseases caused by
PTCs. In Aim 2, I will study the mechanism by which these factors cause stop codon read-through and NMD. I
will use RNA-seq and enhanced ribosome footprint profiling to assess the generality of a factor's role in NMD or
stop codon recognition in vivo and determine mechanistic details about its effect on stop codon recognition.
Mechanistic hypotheses about a protein's effect on translation termination will be tested in a rabbit reticulocyte
lysate translation system to determine how stop codon recognition is affected by these factors. These systems
will enable structural characterization of the interaction of these factors with ribosomes or mRNA by high-
throughput chemical probing and cryo-electron microscopy. Together, these aims will expand our understanding
of the mechanistic underpinnings of premature stop codon recognition, and how this leads to mRNA decay or
stop codon readthrough.
The experiments I propose will advance my technical skills in ribosome biochemistry, human cell culture
techniques, genome-wide CRISPR screening, and mass spectrometry. The latter three of these skillsets will be
particularly important in the development of my own research program and its differentiation from that of my
mentor. The advisory committee that I have assembled, and the accompanying training plan, will aid me in the
development of other essential career skills, including presentation skills, paper and grant writing, and mentoring
and leadership....

## Key facts

- **NIH application ID:** 9869821
- **Project number:** 1K99GM135450-01
- **Recipient organization:** JOHNS HOPKINS UNIVERSITY
- **Principal Investigator:** Boris Zinshteyn
- **Activity code:** K99 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $89,616
- **Award type:** 1
- **Project period:** 2020-02-01 → 2021-01-08

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9869821

## Citation

> US National Institutes of Health, RePORTER application 9869821, Mechanisms for establishment and recognition of stop codon context (1K99GM135450-01). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/9869821. Licensed CC0.

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