A subcellular selective APEX2-based proximity labeling used for identifying mitochondrial G-quadruplex DNA binding...

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• G-quadruplexes (G4s) are non-canonical DNA structures crucial for cellular processes.
• Understanding G4-protein interactions is vital for G4 biology.
• Existing methods lack subcellular spatial specificity for identifying G4 binding proteins (G4BPs).

Purpose of the Study:

• To develop a subcellular selective proximity labeling strategy for investigating mitochondrial DNA (mtDNA) G4 interactomes in living cells.
• To identify novel G4BPs localized within specific subcellular compartments.

Main Methods:

• Utilized an APEX2-based proximity labeling strategy with high spatiotemporal resolution.
• Employed genetically encoded systems for specific labeling within living cells.
• Investigated the biofunctions of identified G4BPs, focusing on DHX30.

Main Results:

• Successfully identified several mtDNA G4BPs using the novel strategy.
• Discovered DHX30 as a previously unrecognized mtDNA G4BP that resolves G4s.
• Demonstrated that DHX30 unfolds mtDNA G4s, decreasing tumor cell glycolysis, an effect reversed by RHPS4.

Conclusions:

• The developed APEX2-based strategy enables the identification of subcellular localized G4BPs with high specificity.
• Provides a novel approach to map protein interactions with nucleic acid structures in specific subcellular compartments.
• Highlights DHX30's role in regulating mtDNA G4s and impacting cellular metabolism.