Probing direct interactions between nuclear proteins in cells with nxReLo.
Sherif Ismail1, Jana Kubíková1, Maria Maichel1
1Heidelberg University Biochemistry Center, Heidelberg University, Heidelberg 69120, Germany.
Summary
We developed nxReLo, a new assay to study nuclear protein-protein interactions (PPIs), crucial for understanding piRNA production and animal reproduction. This method aids in characterizing complex nuclear protein networks.
Area of Science:
- Molecular Biology
- Genetics
- Structural Biology
Background:
- Protein-protein interactions (PPIs) are fundamental to biological processes.
- Studying nuclear protein interactions, especially those involving disordered regions, presents significant challenges.
- Understanding PIWI-interacting RNA (piRNA) pathways is vital for germline genome integrity and animal reproduction.
Purpose of the Study:
- To develop and validate nxReLo, a novel cell culture-based colocalization assay for identifying and characterizing nuclear protein interactions.
- To investigate the protein interaction network of the Rhino-Deadlock-Cutoff (RDC) complex involved in piRNA biogenesis in *Drosophila melanogaster*.
- To combine cell-based assays with structural prediction to gain molecular and structural insights into nuclear protein complexes.
Main Methods:
- Development of nxReLo, a simple, fast, cell culture-based colocalization assay for nuclear PPIs.
- Systematic pairwise interaction screening within the RDC complex and assembly of a four-component complex.
- Integration of nxReLo assays with AlphaFold structural prediction for detailed complex characterization.
- Validation of predicted structures using interface point mutations.
Main Results:
- nxReLo successfully identified and characterized pairwise and multiprotein interactions within the RDC nuclear network.
- Detailed molecular and structural insights were obtained for the Cutoff-Deadlock and Bootlegger-Deadlock complexes.
- Specific interaction domains and critical interface residues were identified and validated through mutation analysis.
- The study demonstrated the utility of nxReLo for structurally complex or challenging nuclear proteins.
Conclusions:
- nxReLo provides a rapid, simple, and effective method for testing direct nuclear protein interactions in a cellular context.
- The assay is particularly valuable for studying structurally challenging proteins or when conventional methods fail.
- The findings advance the understanding of the RDC complex and its role in piRNA biogenesis.
- nxReLo facilitates the study of essential PPIs in fundamental biological processes like germline genome maintenance.
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