RNA tools for probing spliceosome dynamics

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.