# Regulation and biological functions of mRNA Alternative Polyadenylation in the Brain

> **NIH NIH R01** · UNIVERSITY OF PITTSBURGH AT PITTSBURGH · 2020 · $331,141

## Abstract

Project Summary
Alternative polyadenylation (APA) generates highly diverse transcriptome and proteome in the brain to
maintain proper neurological functions. Recent works from others and us revealed a complex and dynamic
APA landscape in the brain with many cell type-specific APA regulations and an emerging role of APA in
neurological disease. However, APA alterations in neurological disorders remain largely unexplored and
rigorously designed in vivo high-throughput APA studies in brain disease are especially lacking due to
technical difficulties. We broke the technical barrier in 2017 by developing the cTag-PAPERCLIP technology,
which simultaneously profiles more than 20,000 poly(A) sites in individual cell types from intact mouse brain
and preserves the in vivo transcriptome better than other sequencing strategies that require cell purification.
Tuberous sclerosis (TSC) is characterized by activation of the mTORC1 signaling in the brain with severe
neurological symptoms. Interestingly, mTORC1 activation was recently shown to cause global proximal
APA shift through regulating an APA factor in non-brain cells, which suggests that aberrant APA is a
previously unknown cellular phenotype of TSC. In this application, we propose to conduct a proof-of-
principle study in which we apply cTag-PAPERCLIP to investigate APA in TSC by incorporating new tools
that we recently developed. We will identify changes in APA isoform expression in response to acute loss of
Tsc1 and activation of mTORC1 signaling by in vivo cTag-PAPERCLIP profiling in 3 brain cell types:
excitatory neurons, inhibitory neurons and astrocytes. We will use our established bioinformatics pipeline to
identify robust and biologically important APA changes in each cell type. We will perform RNA-seq to
provide independent validations for the observed APA changes. APA can alter the cellular signaling network
through production of truncated proteins or loss of 3′ UTR regulation from RNA-binding proteins and
microRNAs. For APA changes predicted to carry such effects, we will experimentally validate the predicted
quantitative or qualitative protein changes in common cell lines and primary neuron cultures. Lastly, we will
also investigate the molecular mechanisms responsible for APA changes from mTORC1 activation in
primary neuron cultures. Upon completion of the proposed project, we expect to reveal for the first time how
mTORC1 activation in TSC alters the cellular APA landscapes in excitatory neurons, inhibitory neurons and
astrocytes in addition to addressing a key question—how APA dysregulation contributes to TSC
pathogenesis. Our results will also provide new insights into the knowledge and clinical management of
TSC and other neurological disorders with mTOR pathway dysregulation (“mTORopathies”)

## Key facts

- **NIH application ID:** 9859570
- **Project number:** 1R01NS113861-01
- **Recipient organization:** UNIVERSITY OF PITTSBURGH AT PITTSBURGH
- **Principal Investigator:** Hun-Way Hwang
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $331,141
- **Award type:** 1
- **Project period:** 2020-03-15 → 2025-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9859570

## Citation

> US National Institutes of Health, RePORTER application 9859570, Regulation and biological functions of mRNA Alternative Polyadenylation in the Brain (1R01NS113861-01). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9859570. Licensed CC0.

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