# The Secret Lives of RNA: The In Vivo 3D-Structural Logic of Single Neuron RNA Metabolism

> **NIH NIH DP1** · UNIVERSITY OF PENNSYLVANIA · 2021 · $1,137,500

## Abstract

RNA metabolism, from its synthesis in the nucleus, through its role in cellular homeostasis, to degradation in
the lysosome, is a regulated process that is inherently controlled by RNA structure. We know this, in part, from
the plethora of papers detailing disease-causing deficiencies in RNA metabolism. Modeling of the “structural
landscape of RNA metabolism” to enable experimental regulation of the process, requires knowledge of what
RNAs are expressed, their 3D-structures, their subcellular location and how they interact with their local
interacting partners. To date, most efforts that generate information about these RNA regulatory processes,
such as RNA interactions with RNA binding proteins, use purified fractions of cellular homogenates from
groups of cells. Such cell-ensemble information is useful, however, the cell-selectivity of these processes and
the dynamics of RNA structural changes across this structural landscape is unknown. The uniqueness of
individual cells and subcellular environments requires that such studies be performed at the level of single
cells. There is currently no experimental approach that allows for structural analysis of RNA molecules across
the RNA metabolic landscape within the natural microenvironment of individual cells. We propose to map the
structural landscape of RNA metabolism in single cells, in vivo, by developing a suite of sensitive, high-
resolution molecular approaches that yields a quantitative 3-D map of all RNA-associated structures within
single cells and subregions of these cells. This approach, called In Vivo Structural Analysis mapping, or VISTA
mapping, uses a combination of protein, RNA and organelle markers to direct subcellular function of light-
activated in situ RNA amplifiers, the product of which is RNA-structure sensitive and informative. The structural
analysis of all RNAs and RNA-associated organelles in a single cell will permit a determination the overall logic
of the RNA metabolic landscape within a cell. The goal of the proposed studies is to create and understand the
“Topological Map of Single Cell RNA Metabolism”. This will be accomplished using newly developed VISTA
mapping. Cell-type specific VISTA maps, generated from neurons in their natural microenvironment, will
provide novel insights into and opportunities for manipulating normal cell biology as well as disease-associated
RNAopathies.!
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## Key facts

- **NIH application ID:** 10224810
- **Project number:** 5DP1AA028409-03
- **Recipient organization:** UNIVERSITY OF PENNSYLVANIA
- **Principal Investigator:** JAMES H EBERWINE
- **Activity code:** DP1 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $1,137,500
- **Award type:** 5
- **Project period:** 2019-09-30 → 2024-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10224810

## Citation

> US National Institutes of Health, RePORTER application 10224810, The Secret Lives of RNA: The In Vivo 3D-Structural Logic of Single Neuron RNA Metabolism (5DP1AA028409-03). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10224810. Licensed CC0.

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