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

## TL;DR

This paper provides a detailed protocol to identify proteins that interact with vault RNAs using a method combining RNA purification and mass spectrometry.

## Contribution

The study introduces a novel protocol for uncovering the protein interactome of small non-coding vault RNAs using RNA antisense purification and mass spectrometry.

## Key findings

- A protocol is described for isolating vtRNA-protein complexes using biotinylated antisense DNA probes and UV crosslinking.
- The method allows for mass spectrometry-based analysis of vtRNA interactors in human lung epithelial cells.
- The protocol is adaptable to other small non-coding RNAs and cell types.

## Abstract

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

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.