Identification of Proteins Binding Coding and Non-coding Human RNAs using Protein Microarrays

We describe a refined approach to identify new human RNA-protein interactions. In vitro transcribed labeled RNA is bound to ~9,400 human recombinant proteins spotted on protein microarrays. This approach identified 137 RNA-protein interactions for 10 human coding and non-coding RNAs, including an interaction between Staufen 1 protein and TP53 mRNA that promoted the latter’s stability. RNA hybridization to protein microarrays allows rapid identification of human RNA-protein interactions on a large scale. Sense and antisense strands for 10 RNA transcripts representing protein coding RNAs TP53, HRAS, MYC, BCL2 and non-coding sequences PWRN1, SOX2OT, OCC1, IGF2RNC, lncRBM26 and DLEU1 were in vitro transcribed, labeled with Cy5 and independently hybridized on human protein microarrays. The labeling process was optimized in order to achieve ~ 3 pmol dye per every microgram RNA with average efficacy of 1 dye molecule for approximately every 850 bp RNA to minimally influence RNA native structure and at the same time yield in signal intensities that were readily visualized.