Visualizing protein-RNA interactions inside cells by fluorescence resonance energy transfer

Area of Science:

• Molecular Biology
• Cell Biology
• Biophysics

Background:

• Studying protein-protein interactions in cells is common, but understanding protein-nucleic acid interactions, especially RNA-binding proteins, needs better methods.
• Visualizing these interactions in real-time within living cells remains a challenge.

Purpose of the Study:

• To develop and validate a novel in situ method for visualizing RNA-binding protein-RNA interactions using fluorescence resonance energy transfer (FRET).
• To analyze the spatial distribution and RNA-binding patterns of specific alternative splicing repressors, PTB and Raver1.

Main Methods:

• Developed a FRET-based approach combining a yellow fluorescent protein (YFP)-tagged RNA-binding protein and SytoxOrange-stained RNA.
• Utilized fluorescence lifetime imaging microscopy (FLIM) to measure FRET efficiency, indicated by a decrease in YFP fluorescence lifetime upon RNA binding.
• Applied the method to study the nuclear localization and RNA interactions of PTB and Raver1.

Main Results:

• Successfully demonstrated RNA-specific FRET signals, confirming the method's ability to detect protein-RNA association.
• Observed that PTB and Raver1 bind to RNAs throughout the nucleus.
• Discovered an additional interaction of PTB and Raver1 with RNAs specifically within the perinucleolar compartment (PNC), where noncoding Pol III transcripts are located.

Conclusions:

• The developed FLIM-FRET method provides a powerful tool for studying RNA-binding protein dynamics and localization in situ.
• PTB and Raver1 exhibit distinct RNA-binding patterns, interacting with both coding and noncoding RNAs in the nucleus and perinucleolar compartment.