Protocol to uncover the protein interactome of small non-coding vault RNAs through RNA antisense purification coupled to mass spectrometry

Vault RNAs (vtRNAs) are a family of four small non-coding RNAs (ncRNAs). Here, we present a protocol to identify protein interactors of vtRNA1-1, vtRNA1-2, and vtRNA1-3 in human lung epithelial cells through RNA antisense purification coupled to mass spectrometry (RAP-MS). We describe steps for using biotinylated vtRNA-specific antisense DNA probes to isolate vtRNA-protein complexes that are covalently crosslinked by treatment with ultraviolet (UV) light, followed by mass spectrometry-based analysis. This protocol can be adapted to other cells or small ncRNAs. For complete details on the use and execution of this protocol, please refer to Stok et al. 1 • Steps to identify protein interactome of vault RNAs during infection with EMC virus • Guidelines on how to design an optimal probe for targeting small non-coding RNAs • Steps for performing RNA antisense purification coupled to mass spectrometry • Guidance on how to interpret and analyze the obtained mass spectrometry data Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Vault RNAs (vtRNAs) are a family of four small non-coding RNAs (ncRNAs). Here, we present a protocol to identify protein interactors of vtRNA1-1, vtRNA1-2, and vtRNA1-3 in human lung epithelial cells through RNA antisense purification coupled to mass spectrometry (RAP-MS). We describe steps for using biotinylated vtRNA-specific antisense DNA probes to isolate vtRNA-protein complexes that are covalently crosslinked by treatment with ultraviolet (UV) light, followed by mass spectrometry-based analysis. This protocol can be adapted to other cells or small ncRNAs.