# Systematic characterization of trans regulation of A-to-I RNA editing in neurons

> **NIH NIH R01** · STANFORD UNIVERSITY · 2020 · $454,121

## Abstract

Project Summary/Abstract
RNA editing is a critical process for generating spatiotemporal transcriptomic diversity that is
particularly important in the brain. A-to-I (adenosine to inosine, which is recognized as
guanosine) editing is the most common form of RNA editing in metazoans and is catalyzed by
a family of enzymes called adenosine deaminases acting on RNA (ADARs). A-to-I editing
occurs co-transcriptionally when double-stranded RNA (dsRNA) is bound and edited by
ADAR enzymes, which occurs at high frequency in the nervous system. Alteration of RNA
editing levels is implicated in a number of neurological disorders. Loss of ADAR can lead to
neurological phenotypes such as seizure, altered locomotion and circadian rhythm. Previous
work studying mutations in ADAR demonstrates the importance of a few amino acids critical
for proper editing activity, including a handful known to cause human diseases. However, we
still lack a comprehensive understanding of ADAR protein function. We know even less about
other trans regulators of RNA editing despite the evidence suggesting their existence. In this
work, we aim to develop systematic approaches to deciphering the trans regulation of A-to-I
RNA editing. First, we will identify functional mutants of ADAR1 and ADAR2 in human cells.
Using a CRISPR-based technology we recently developed, we will perform saturation
mutagenesis of ADAR1 and ADAR2 to introduce point mutations in human cells. We will
identify functional ADAR mutants with decreased or increased editing activity and further
characterize how these mutations affect ADAR editing activity in vivo. Second, we will
identify novel regulators of RNA editing through biochemical and genetic screens. We will
identify ADAR-interacting proteins in induced human neurons. We will also carry out a
genome-wide CRISPR/Cas9 screen in induced human neurons to find candidates that alter
editing levels. Top candidate genes are subject to secondary CRISPR/Cas9 screening in
mouse primary neurons as well as double knockout in pairwise combinations to analyze their
genetic interactions. Third, we will determine mechanistically how editing regulators alter the
transcriptome-wide landscape of RNA editing. We will perturb the regulators to examine how
they affect editing levels transcriptome-wide in human cells, mouse primary neurons, and
Drosophila brains. We will test whether the regulators physically interact with ADAR1/2 or
each other, and if and how they interact with ADAR RNA substrates. This work will provide
an unprecedented understanding of trans regulation of A-to-I RNA editing in neurons,
revealing novel mechanisms underlying this largely unexplored machinery.

## Key facts

- **NIH application ID:** 9974571
- **Project number:** 5R01MH115080-04
- **Recipient organization:** STANFORD UNIVERSITY
- **Principal Investigator:** Jin Billy Li
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $454,121
- **Award type:** 5
- **Project period:** 2017-09-15 → 2022-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9974571

## Citation

> US National Institutes of Health, RePORTER application 9974571, Systematic characterization of trans regulation of A-to-I RNA editing in neurons (5R01MH115080-04). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9974571. Licensed CC0.

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