Spatial mapping of RNA-microenvironments with HCR-Proxy
The spatial organisation of biomolecules enables cells to efficiently regulate numerous biological processes. In addition to canonical membrane bound organelles, biomolecular condensates represent an important organisational unit, usually formed by weak interactions between RNA molecules and RNA-binding proteins. Even minor changes in a condensate’s interaction network can disrupt cellular processes and, consequently, lead to the development of various diseases. Despite numerous approaches for studying protein interactors of target RNAs, there remains a need to develop a method capable of spatially resolving interaction partners within multi-layered condensates.
In the published work, researchers from the Laboratory for RNA Networks developed a new RNA-centric proximity labelling method called HCR-Proxy. Method combines a signal amplification technique (HCR, Hybridisation Chain Reaction) with an in situ proximity labelling approach (Proxy). The main advantage of the method is probe-independent labelling of RNA’s surroundings, which is especially important in the context of studying problematic RNA targets. In the collaboration with researchers from Jožef Stefan Institute method was technically refined, where with the implementation of STED microscopy spatial resolution of labelling was improved. The researchers successfully applied the HCR-Proxy method to a multi-layered condensate, the nucleolus, where they identified protein interactors specific for each nucleolar sublayer. Further application of machine learning also enabled prediction of key protein features such as protein charge, molecular size and the presence of functional domains, suggesting their contribution in governing protein distribution across nucleolar sublayers.
Therefore, HCR-Proxy presents a versatile tool with high flexibility, ease of use and wide range of application. Besides addressing condensate’s compositional architecture, HCR-Proxy method can also be used as a screening tool for discovering small molecules capable of modulating condensate's properties for therapeutic purposes.
More information: https://academic.oup.com/nar/article/54/4/gkag086/8494757
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