RNA-protein interaction mapping via MS2- or Cas13-based APEX targeting.

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• RNA-protein interactions are crucial for cellular functions.
• Current methods for mapping these interactions in vivo are limited.
• Systematic and unbiased approaches are needed to identify RNA-binding proteins.

Purpose of the Study:

• To develop and apply novel RNA-centered proximity biotinylation methods.
• To identify novel protein partners interacting with human telomerase RNA (hTR) in living cells.
• To investigate the functional role of identified protein partners in telomerase regulation.

Main Methods:

• Targeting engineered peroxidase APEX2 to specific RNAs using MS2-MCP and CRISPR-Cas13 systems.
• Performing RNA-centered proximity biotinylation for one minute.
• Identifying biotinylated proteins via mass spectrometry.
• Validating protein-RNA interactions and functional consequences.

Main Results:

• Successfully targeted APEX2 to hTR with high specificity using both MS2-MCP and CRISPR-Cas13 systems.
• Identified over a dozen novel candidate binding partners for hTR.
• Validated the interaction between hTR and ALKBH5, an m6A demethylase.
• Demonstrated that ALKBH5 modifies endogenous hTR and affects telomerase activity.

Conclusions:

• MS2- and Cas13-targeted APEX2 are effective tools for unbiased discovery of RNA-protein interactions in living cells.
• ALKBH5 is a novel interactor of hTR that plays a role in telomerase regulation.
• These methods open new avenues for studying RNA biology and identifying novel therapeutic targets.