Capture and Purification of Endogenous Long Noncoding Ribonucleic Protein Complexes for Protein Identification Using Grad-MS.

The composition of long noncoding ribonucleic protein (lncRNPs) complexes has a major impact on lncRNA function, revealing a multitude of modalities for RNA-protein interactions. However, long noncoding RNAs (lncRNAs) also interact with an array of proteins during their processing that are not related to their function. For example, different proteins involved in splicing, maturation, and nuclear export are likely to interact with a newly transcribed lncRNA. Thus, a major limitation is the purification of fully complexed lncRNPs after the transient, intermediate RNA-protein interactions. Here we outline a newly developed protocol called Sucrose Gradient coupled to Mass Spectrometry (Grad-MS): a lncRNA-centric approach to enrich lncRNPs of specified sedimentation value-indicative of fully complexed lncRNP-and identify their associated proteins. RNA-integrated complexes are cross-linked in vivo and fractionated in a sucrose gradient. Then, target lncRNAs are purified from the specific sedimentation fraction using biotinylated tiling antisense oligos, followed by protein detection using mass-spectrometry. We further outline the steps using Grad-MS to isolate discrete lncRNPs in the cytoplasm (e.g., Norad) and nucleus (e.g., Firre). Overall, Grad-MS allows the enrichment and purification of lncRNPs to facilitate protein identification.