A versatile assay for RNA-binding proteins in living cells

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• RNA-binding proteins (RBPs) regulate RNA metabolism, but their in vivo activity is difficult to measure.
• Existing methods like EMSA provide indirect measures of RBP activity in cells.
• Accurate assessment of RBP-mRNA interactions is crucial for understanding gene regulation.

Purpose of the Study:

• To develop a quantitative in vivo method for assessing RNA-binding activity of RBPs.
• To enable monitoring of dynamic changes in RBP activity in response to stimuli.
• To facilitate high-throughput screening of RBP-RNA interactions.

Main Methods:

• Fusion of RBPs to fluorescent proteins (e.g., eGFP).
• In vivo cross-linking of protein-RNA complexes using UV irradiation.
• Immunoprecipitation using a GFP-binding protein (GBP) and detection of bound polyadenylated RNA via oligo(DT) hybridization with a red fluorophore.

Main Results:

• A quantitative dual fluorescence assay for in vivo RBP-RNA interaction analysis was established.
• The method allows monitoring of dynamic changes in RNA-binding activity.
• Adaptation for 96-well plates enables high-throughput RNA-binding measurements.

Conclusions:

• This novel, fast, and radioactivity-free method provides a direct measure of RBP activity in vivo.
• The assay facilitates high-throughput screening, aiding RNA biology research.
• It offers a valuable tool for studying RBP function and regulation.