# High-throughput dissection of RNA localization regulatory elements

> **NIH NIH F31** · UNIVERSITY OF COLORADO DENVER · 2023 · $36,354

## Abstract

PROJECT SUMMARY/ ABSTRACT
RNA localization is critical for a diverse set of biological processes. The localization of an RNA depends on
cis-elements, features inherent to the transcript, and trans-factors, effectors independent of the target
transcript, which are often RNA binding proteins. Cis-elements that regulate RNA localization, often called
"zip-codes," are often found in the 3′ untranslated regions (UTRs) of transcripts. However, for the more than
99% of the known localized RNAs, the cis-elements that regulate their localization are unknown. Recent work
from the Taliaferro lab identified several 260 nucleotide RNA sequences within the 3’ UTRs of some
neurite-localized RNAs that were necessary and sufficient for neurite RNA transport. These were identified
using a massively parallel reporter assay that screened approximately 10,000 RNA sequences drawn from
endogenous 3’ UTRs for their ability to traffick a reporter transcript to neurites. Interestingly, 100 nt
subsequences of these 260 nt active elements were not capable of directing RNA transport. This indicates that
(1) the minimal regulatory elements are quite large (likely longer than 100 nt) and (2) the true character of the
localization regulatory elements remains unknown.
In this work, it is proposed to comprehensively characterize the previously identified RNA localization
regulatory elements and zero in on their important features. This will be done by generating a pool of 10,000
RNA sequences based on the previously identified 260 nt zipcodes. Each sequence in this pool will contain
defined deletions of varying sizes that span the length of the zipcode. By integrating these mutants into the 3’
UTR of a reporter transcript assaying which of them retain the ability to direct localization of the reporter to
neurites, a quantitative readout of the functional importance of each nucleotide within the 260 nt zipcode will be
obtained. From this, a clear picture of the important features that make up active localization elements will
arise, facilitating their identification in other localized RNAs.
The large size of these zipcodes suggests that their secondary may be important for their activity. To test this,
their secondary structure will be determined using chemical probing techniques. To test the functionality of the
structure, thousands of mutants that disrupt RNA structure as well as compensatory mutants that restore it will
be generated. As above, the ability of each of these mutants to drive a reporter transcript to neurites will be
tested. In this way, RNA structure and function will be directly related.
Answering these questions will help in understanding the underlying mechanisms of RNA localization as very
few examples of RNA localization have known mechanistic underpinnings. Identifying the mechanism of RNA
localization under physiological conditions is important to being able to understand potential dysfunction in
disease states.

## Key facts

- **NIH application ID:** 10749328
- **Project number:** 1F31GM151819-01
- **Recipient organization:** UNIVERSITY OF COLORADO DENVER
- **Principal Investigator:** Charlie E Moffatt
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $36,354
- **Award type:** 1
- **Project period:** 2023-08-01 → 2025-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10749328

## Citation

> US National Institutes of Health, RePORTER application 10749328, High-throughput dissection of RNA localization regulatory elements (1F31GM151819-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10749328. Licensed CC0.

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