HyPro-MS analysis of proteins proximal to nuclear noncoding RNAs in HeLa cells

The ability of RNAs to form specific contacts with other macromolecules provides an important mechanism for subcellular compartmentalization. We developed a suite of hybridization-proximity (HyPro) labeling technologies for unbiased discovery of proteins (HyPro-MS) and transcripts (HyPro-seq) associated with RNAs of interest in genetically unperturbed cells. To generate the HyPro-MS dataset reported here, fixed and permeabilized HeLa cells were hybridized with digoxigenin-labeled oligonucleotide probes against noncoding RNAs 45S, NEAT1 or PNCTR and proteins co-localizing with these RNAs were biotinylated in situ using a custom-engineered HyPro enzyme containing a digoxigenin-binding domain. Biotinylated proteins were then captured on streptavidin beads and analyzed by LC-MS/MS.