Post-transcriptional regulation of gene expression is an important process that is mediated by interactions between mRNAs and RNA binding proteins (RBP), non-coding RNAs (ncRNA) or ribonucleoproteins (RNP). Key to the study of post-transcriptional regulation of mRNAs and the function of ncRNAs such as long non-coding RNAs (lncRNAs) is an understanding of what factors are interacting with these transcripts. While several techniques exist for the enrichment of a transcript whether it is an mRNA or an ncRNA, many of these techniques are cumbersome or limited in their application.
Here researchers from the Washington University School of Medicine present a novel method for the immunoprecipitation of mRNAs and ncRNAs, Urb—RNA immunoprecipitation (Urb-RIP). This method employs the RRM1 domain of the “resurrected” snRNA-binding protein Urb to enrich messages containing a stem-loop tag. Unlike techniques which employ the MS2 protein, which require large repeats of the MS2 binding element, Urb-RIP requires only one stem-loop. This method routinely provides over ~100-fold enrichment of tagged messages. Using this technique the researchers have shown enrichment of tagged mRNAs and lncRNAs as well as miRNAs and RNA-binding proteins bound to those messages. They have confirmed, using Urb-RIP, interaction between RNA PolIII transcribed lncRNA BC200 and polyA binding protein.
Schematic illustration of the Urb-RIP protocol and potential applications
The first step of the Urb-RIP protocol is to tag an RNA of interest with the SLII-tag, illustrated in the figure. The tagged RNA and in parallel an untagged control are coexpressed with 2HA-Urb in a cell line of interest. After a period of time the cells are UV irradiated to produce RNA-protein crosslinks. The cells are then lysed and immunoprecipitation is performed using blocked anti-HA magnetic beads. The RNA or protein is then eluted and analyzed by an appropriate method. This approach should be applicable to mRNAs and lncRNAs and amendable to a wide range of methods for eluate analysis.