PRIMA: a gene-centered, RNA-to-protein method for mapping RNA-protein interactions

ABSTRACT

Interactions between RNA binding proteins (RBPs) and mRNAs are critical to post-transcriptional gene regulation. Eukaryotic genomes encode thousands of mRNAs and hundreds of RBPs. However, in contrast to interactions between transcription factors (TFs) and DNA, the interactome between RBPs and RNA has been explored for only a small number of proteins and RNAs. This is largely because the focus has been on using ‘protein-centered’ (RBP-to-RNA) interaction mapping methods that identify the RNAs with which an individual RBP interacts. While powerful, these methods cannot as of yet be applied to the entire RBPome. Moreover, it may be desirable for a researcher to identify the repertoire of RBPs that can interact with an mRNA of interest—in a ‘gene-centered’ manner—yet few such techniques are available. Here, we present Protein-RNA Interaction Mapping Assay (PRIMA) with which an RNA ‘bait’ can be tested versus multiple RBP ‘preys’ in a single experiment. PRIMA is a translation-based assay that examines interactions in the yeast cytoplasm, the cellular location of mRNA translation. We show that PRIMA can be used with small RNA elements, as well as with full-length Caenorhabditis elegans 3′ UTRs. PRIMA faithfully recapitulated numerous well-characterized RNA-RBP interactions and also identified novel interactions, some of which were confirmed in vivo. We envision that PRIMA will provide a complementary tool to expand the depth and scale with which the RNA-RBP interactome can be explored.

KEYWORDS:

• 3′ untranslated region
• caenorhabditis elegans
• RNA
• RNA binding protein
• yeast

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgments

The authors would like to thank Allan Jacobson, Job Dekker and members from the Walhout laboratory for advice and critical reading of the manuscript. Additionally, the authors would like to thank Allan Jacobson, Marvin Wickens and Paul Kaufman for reagents, and Phil Zamore and Nick Rhind for access to equipment.

Funding

This work was supported by the National Institutes of Health under Grant HG006234 to A.J.M.W. and under Grant GM117237 to S.P.R.