RiboPuromycylation Method (RPM): A New Technique for Visualizing Active Cellular Translation Sites and Exploring Translation Compartmentalization

Evidence is mounting that regulating protein synthesis extends beyond quantitative control of translation of bulk or individual mRNAs. The localization of polysomes, which is clearly non‐random, probably plays a key role in many cellular processes, including migration, polarization & synapse formation through the local synthesis of proteins that participate in these processes. Existing methods to visualize cellular translation sites (isotopic labels, fluorescent puromycin) suffer from poor sensitivity, compatibility with imaging other fluors, and linearity. We developed the RiboPuromyclation method (RPM) to visualize and quantify actively translating ribosomes in cells by standard immunofluorescence. RPM is based on the selective puromycylation of nascent chains stably bound to translating ribosomes and detection of the puromycylated nascent chains using a puromycin‐specific monoclonal antibody. We provide extensive biochemical evidence supporting this conclusion, including the initial description of an ELISA to analyze binding of chemicals/proteins to native ribosomes/polysomes. Research Support: NIH