Biochemical Pulldown of mRNAs and Long Noncoding RNAs from Cellular Lysates Coupled with Mass Spectrometry to Identify Protein Binding Partners.

Abstract

RNA binding proteins (RBPs) interact with cellular mRNAs, controlling various steps throughout the lifetime of these transcripts, including transcription, cellular transport, subcellular localization, translation and degradation. In addition to binding mRNA transcripts, a growing number of RBPs are shown to bind long noncoding RNAs (lncRNAs), controlling key cellular processes, including gene expression and translation of proteins. Current methodologies aimed at identifying and characterizing protein binding partners of specific RNAs of interest typically rely on tagging of the RNA with affinity aptamers, using in vitro transcribed RNA or immobilized oligonucleotides to capture RNA-protein complexes under native conditions. These assays are coupled with mass spectrometry or Western Blot analysis to identify or/and confirm interacting proteins. Here, we describe an alternative approach to identify protein binding partners of mRNAs and large long noncoding RNAs. This approach relies on biochemical pulldown of specific target RNAs and interacting protein partners from cellular lysates coupled with mass spectrometry to identify novel interacting proteins. By using 24-48 ~20 mer biotinylated DNA probes that hybridize to the target RNA, the method ensures high specificity and minimal off target binding. This approach is reproducible and fast and serves as a base for discovery studies to identify proteins that bind to RNAs of interest.