An Aptamer-based mRNA Affinity Purification Procedure (RaPID) for the Identification of Associated RNAs (RaPID-seq) and Proteins (RaPID-MS) in Yeast.

ABSTRACT

RNA-RNA and RNA-protein interactions are involved in the regulation of gene expression. Here, we describe an updated and extended version of our RNA purification and protein identification (RaPID) protocol for the pulldown of aptamer-tagged mRNAs by affinity purification. The method takes advantage of the high affinity interaction between the MS2 RNA aptamer and the MS2 coat protein (MCP), as well as that between streptavidin-binding peptide (SBP) and streptavidin. Thus, it employs MCP-SBP fusions to affinity purify MS2-tagged target RNAs of interest over immobilized streptavidin. Purified aptamer-tagged mRNAs, along with any associated RNAs and proteins, are then sent for RNA sequencing (RaPID-seq) or mass spectrometry (RaPID-MS), which allows for the identification of bound cohort RNAs and proteins, respectively.