Targeted cross-linking-mass spectrometry determines vicinal interactomes within heterogeneous RNP complexes

Area of Science:

• Molecular Biology
• Biochemistry
• Proteomics

Background:

• Ribonucleoprotein particles (RNPs) are crucial cellular components, but their internal organization and dynamic protein interactions remain poorly understood.
• Existing proteomic and RNomic methods identify RNP components but offer limited insights into spatial organization and transient interactions.

Purpose of the Study:

• To develop and validate a targeted cross-linking approach to map protein proximities within dynamic RNPs.
• To investigate the changing vicinity interactomes of RNPs during maturation pathways.

Main Methods:

• A targeted cross-linking strategy combining a known anchor site, affinity purification, and mass spectrometry (MS).
• Utilizing a heterobifunctional cross-linker with a confined reaction radius to capture transient and low-abundance protein interactions.
• Application to Saccharomyces cerevisiae mRNA export receptor Mex67:Mtr2 and pre-ribosomal Nop7 subcomplex.

Main Results:

• Successfully identified dynamic vicinal interactomes within the studied RNP complexes.
• Captured transient and low-abundance protein interactions previously difficult to detect with traditional methods.
• Demonstrated the method's efficacy in mapping protein proximities along RNP maturation pathways.

Conclusions:

• The developed targeted cross-linking method provides a novel tool for studying the spatial organization of heterogeneous and dynamic RNP complexes.
• This approach enhances our understanding of RNP assembly and function by revealing dynamic protein proximities.
• The findings offer new avenues for investigating molecular mechanisms within ribonucleoprotein particles.