# Defining the Boundaries of RNAi in a cell

> **NIH NIH F31** · UT SOUTHWESTERN MEDICAL CENTER · 2022 · $30,752

## Abstract

Project Summary
 RNA interference (RNAi) has driven a surge of research over the past twenty years as a powerful
tool for gene silencing, and the discovery of a distinct class of noncoding RNAs, microRNAs (miRNAs),
has revolutionized our outlook of genome regulation. miRNAs bind to a critical effector molecule, the
Argonaute protein, to achieve sequence-specific recognition and repression of their target. Because
the rules for complementary recognition seem simple and predictable, the dysregulation of miRNAs
has been implicated as the sole culprit of disease in over 40,000 articles on PubMed. The sheer volume
of publications suggest that miRNAs have omnipotent control over gene expression. This model of
ubiquitous miRNA regulation suggested that there is an urgent need for a fundamental reexamination
of the available gene silencing infrastructure present in a cell.
 Progress in identifying high-quality candidates for miRNA-based therapies have been hindered
by this major barrier that miRNAs are expected to recognize more targets in a cell than there are
catalytic molecules to guide. This project seeks to answer the question of how the competition for
shared gene silencing resources can have a dramatic impact on the number of targets miRNAs can
control. The first and second aim will both use quantitative mass spectrometry (MS) to count the AGO
molecules per cell and RNA-sequencing to determine the miRNA and mRNA expression in a HCT116
colon cancer model system. While the first aim will interrogate CRISPR-mediated AGO knockout cell
lines, the second aim will provide the complementary overexpression analysis. Furthermore, these cell
lines will be used as rigorous controls for unbiased identification of the AGOs’ respective RNA binding
sites using AGO-eCLIP-Seq. The third aim is to use three types of cell stress, nutrient deprivation,
oxidative stress, and hypoxia, to trigger an endogenous RNAi response, which will be assessed by
AGO copy number with quantitative MS, miRNA and target expression with RNA-Seq techniques, and
RNA binding locations with AGO-eCLIP-Seq.
 The multi-disciplinary environment in the Corey laboratory fosters collaboration amongst diverse
backgrounds, and trainees benefit from Dr. Corey’s attentive mentorship and the guidance of several
established research scientists. By the end of this training fellowship period, I will accomplish four goals:
1) to be knowledgeable about the literature in my chosen field of RNA biology, 2) to be comfortable
writing grant proposals and manuscripts, 3) to be technically competent, and 4) speak with clarity to all
audiences.

## Key facts

- **NIH application ID:** 10396103
- **Project number:** 5F31GM137591-03
- **Recipient organization:** UT SOUTHWESTERN MEDICAL CENTER
- **Principal Investigator:** Krystal Courtney Johnson
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $30,752
- **Award type:** 5
- **Project period:** 2020-05-11 → 2023-05-10

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10396103

## Citation

> US National Institutes of Health, RePORTER application 10396103, Defining the Boundaries of RNAi in a cell (5F31GM137591-03). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10396103. Licensed CC0.

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