Fluorescent shift assay for APOBEC-mediated RNA editing.

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Cytidine (C) to Uridine (U) RNA editing is a crucial post-transcriptional modification impacting various biological processes.
• The APOBEC deaminase family is known to induce C-to-U mutations in RNA and DNA, playing roles in cellular functions.
• Directly comparing the activity of different RNA editing enzymes is challenging due to a lack of efficient readout systems.

Purpose of the Study:

• To develop and detail a novel system for quantifying RNA editing activity.
• To enable direct and efficient comparison of RNA editing enzyme efficiencies.
• To provide a flexible tool for studying RNA editing in cellular contexts.

Main Methods:

• A modified enhanced green fluorescent protein (eGFP) construct was engineered to change subcellular localization upon RNA editing.
• RNA editing levels were assessed by measuring the ratio of nuclear to cytosolic eGFP fluorescence intensity using confocal microscopy.
• DNA-binding stains like DAPI or Hoechst were used to identify the cellular nucleus for accurate intensity ratio calculations.

Main Results:

• The developed system allows RNA editing to directly influence the subcellular localization of the modified eGFP.
• Relative fluorescence intensity can be accurately compared based on the measured RNA editing levels.
• The system facilitates the quantification of RNA editing activity within individual cells.

Conclusions:

• This novel system provides a powerful and adaptable tool for examining and quantifying RNA editing activity.
• The method is applicable beyond APOBEC proteins, extending to general RNA editing enzymatic assays.
• It overcomes limitations of previous systems by offering a direct and efficient readout for RNA editing enzymes.