# RNA tools for probing spliceosome dynamics

> **NIH NIH R00** · GEORGETOWN UNIVERSITY · 2022 · $243,080

## Abstract

Project Summary/Abstract
 Alternative splicing is a central mechanism to diversify genetic information on the post-transcriptional level.
Advances in sequencing technologies revealed shifts in alternative splicing patterns as key features in a variety
of biologically relevant systems including embryo development, the adaptive immune response and cancer
progression. A recent RNAseq study demonstrated that alternative splicing patterns for thousands of transcripts
are altered in macrophages infected with Listeria. While proteins and mechanisms involved are not established,
a protective cellular response to limit intracellular replication may be a consequence. The central goal of this
proposal is to use this infection model system to gain insights into dynamics of non-coding RNAs and
mechanisms of alternative splicing on a single cell level. Intriguingly, it was independently discovered that
spliceosome components are transiently sequestered in cytosolic RNA-protein granules called U-bodies during
Listeria infection, suggesting that spatiotemporal sequestration may contribute to alternative splicing regulation.
Infection with Listeria and formation of U-bodies are highly heterogeneous both in space and time and ideally
must be assessed on a single-cell basis. Fluorescence microscopy offers the possibility for long-term
visualization of tagged proteins and fluorescently labeled pathogens, but robust tools to visualize cellular RNAs
are limiting. To enable visualization of non-coding RNAs, a versatile tool to fluorescently label RNA in live cells
will be developed (Aim 1). This tool will then be utilized to quantify spatiotemporal dynamics of U-bodies and
simultaneously monitor Listeria replication (Aim 2). Contributions of spliceosome components will be dissected
by monitoring RNA dynamics and Listeria replication as spliceosome components will be manipulated
experimentally. Lastly, a time resolved quantitative mass spectrometry approach will be used to identify protein
candidates that regulate re-shaping of the alternative splicing landscape (Aim 3). These candidate factors will
be further investigated by knockdown and assessing consequences for U-body dynamics and intracellular
bacterial replication in the microscopy assay. Together, this study will serve as a unique model system to unravel
alternative splicing regulation on a single cell level in a physiologically relevant model system using fluorescence
microscopy.

## Key facts

- **NIH application ID:** 10328275
- **Project number:** 5R00GM127752-04
- **Recipient organization:** GEORGETOWN UNIVERSITY
- **Principal Investigator:** Esther Braselmann
- **Activity code:** R00 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $243,080
- **Award type:** 5
- **Project period:** 2021-01-13 → 2023-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10328275

## Citation

> US National Institutes of Health, RePORTER application 10328275, RNA tools for probing spliceosome dynamics (5R00GM127752-04). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10328275. Licensed CC0.

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