Dynamic visualization of mRNA splicing variants with a transactivating reporter

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Pre-mRNA splicing is crucial for gene expression, but dynamic changes like intron loss/gain are difficult to monitor non-invasively.
• Current methods lack the sensitivity to track splicing dynamics in real-time within living systems.

Purpose of the Study:

• To develop a novel reporter system for real-time imaging and monitoring of pre-mRNA splicing dynamics.
• To enable high-throughput screening of compounds that modulate splicing activity.

Main Methods:

• Designed a two-step transcriptional activation (TSTA) reporter system.
• Utilized the GAL4-VP16 fusion protein to target upstream activation sequence (UAS) elements.
• Measured firefly luciferase reporter gene expression to quantify splicing activity.

Main Results:

• Successfully achieved real-time imaging of pre-mRNA splicing in living cells and animals.
• Consistently detected dynamic splicing activity in response to exogenous splicing modulators.
• Demonstrated the reporter's efficacy in tracking splicing changes.

Conclusions:

• The TSTA reporter is a valuable tool for non-invasive, real-time monitoring of splicing.
• This system facilitates high-throughput screening of splicing modulators.
• Accelerates the development of therapeutics for splicing-related disorders.