# SMALL NON-CODING RNA REGULATION OF RAS-GTPase FUNCTION  IN EPIDERMAL HOMEOSTASIS

> **NIH NIH K01** · STANFORD UNIVERSITY · 2021 · $127,386

## Abstract

SMALL NON-CODING RNA REGULATION OF RAS-GTPase FUNCTION in EPIDERMAL HOMEOSTASIS
PROJECT SUMMARY/ABSTRACT
 The Ras-MAPK signal transduction pathway is a critical regulator of the epidermis as dysregulation of Ras-
MAPK signaling inhibits epidermal differentiation and is a major driver of tumorigenesis. Our recent discovery
that snoRNAs directly interact with and regulate Ras function represents a major paradigm shift in our
understanding of small GTPase regulation. Using our novel UV-C cross-linking and immunoprecipitation
platform, irCLIP, to characterize transcriptome wide RAS-superfamily GTPase interactions with RNA, we have
discovered a rich and complex web of snoRNA-RAS-GTPase interactions suggesting that snoRNAs may
regulate all biological processes under RAS-superfamily control, including biochemical signaling nodes,
actin/membrane organization, vesicular and intracellular protein trafficking and nuclear/cytoplasmic transport.
The long term goals of this K01 application are to deeply characterize the regulatory functions and
mechanisms of action of small nucelolar RNAs in modulation of Ras and RAS-superfamily GTPases in control
of epidermal homeostasis.
 In Aim I, we will focus on defining the specificity and breadth of C/D box snoRNA modulation of RAS-
superfamily GTPase functions. Our preliminary irCLIP-seq data showed that members of all 5 RAS-subfamilies,
RAS, RHO, ARF, RAB and RAN, directly interacted with SNORD50A/B. Thus SNORD50A/B may be a global
repressor of RAS-superfamily GTPases as has been described for K-Ras. Using CRISPR/Cas9 gene editing,
SNORD50A/B loss-of-function studies will test RAS-GTPase activation levels of 9 RAS-superfamily GTPases
spanning all 5 subfamilies. Activation status of biochemical pathways downstream of active-RAS-GTPases will
also be monitored with IP-kinase assays and/or phospho-immunoblots when applicable. Our irCLIP-seq data
also revealed that Ras isoforms interacted with >20 C/D box snoRNAs, several of which are amplified in
cancer. This supports the hypothesis that multiple snoRNAs participate in the regulation of Ras function. In Aim
IB, we will use CRISPR-mediated gene editing to excise select Ras-interacting snoRNAs from primary human
keratinocytes and assess loss-of-function via analysis of Ras-GTP levels, ERK1/2 and AKT phosphorylation
levels, and on epidermal homeostasis in 3D human tissue models. Together, this aim will reveal the extent to
which C/D box snoRNAs regulate Ras and RAS-superfamily GTPase functions.
 Aim II is designed to functionally characterize the RNA-dependent Ras protein interactome. Because of
their ability to suppress interaction of Ras with farnesyltransferase, we hypothesized that SNORD50A/B
function as adaptors to modulate specific Ras-protein interactions. Using a tandem affinity purification and
proximal protein biotinylation (BioID) approach, we compared the interactomes of WT to mutant Ras against
WT Ras in a SNORD50A/B +/+ or -/- background. This led to a distille...

## Key facts

- **NIH application ID:** 10136526
- **Project number:** 5K01AR071481-05
- **Recipient organization:** STANFORD UNIVERSITY
- **Principal Investigator:** Brian J Zarnegar
- **Activity code:** K01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $127,386
- **Award type:** 5
- **Project period:** 2017-06-01 → 2022-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10136526

## Citation

> US National Institutes of Health, RePORTER application 10136526, SMALL NON-CODING RNA REGULATION OF RAS-GTPase FUNCTION  IN EPIDERMAL HOMEOSTASIS (5K01AR071481-05). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10136526. Licensed CC0.

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