AKAP12 UV RNA immunoprecipitation followed by high throughput sequencing

Regulation of subcellular mRNA localisation is a fundamental biological mechanism, which adds a spatial dimension to the diverse layers of post-transcriptional control of gene expression. Insights into this phenomenon have been described in a multitude of cell types and across taxonomic kingdoms, highlighting its biological significance. The cellular compartment in which mRNAs are located may define distinct aspects of the encoded proteins, ranging from production rate and complex formation to localised activity. Ultimately, mRNA localisation supports compartmentalised mechanistic outputs that can respond to local stimuli, orient migration, or even shape cells. We used an unbiased method to profile the RNA-bound proteome in migrating endothelial cells and found that the plasma membrane (PM)-associated A-kinase anchor protein 12 (AKAP12) interacts with various mRNAs, including mRNAs encoding kinases with Actin remodelling activity. To identify transcripts bound by AKAP12, we carried out UV crosslinking experiments followed by RNA immunoprecipitation and high throughput sequencing. Our data offer novel insights in complex mechanisms of spatial control of gene expression. Overall design: Human umbilical vein endothelial cells (HUVECs) were grown to 90% confluency in complete endothelial growth media, exposed to 254 nm UV light and lysed. Cell lysates were incubated with an antibody targeting AKAP12 and 5% saved for Input sequencing. Next, antibody-AKAP12 complexes were recovered with G protein magnetic beads. After washing, the complexes were eluted from beads, treated with a revere crosslink buffer containing Proteinase K and the RNA recovered via Trizol extraction.