Polysomes identified by live imaging of nascent peptides are stalled in hippocampal and cortical neurites

Abstract

In neurons, mRNAs can be repressed postinitiation and assembled into granules enabling the transport and later, regulated reactivation of the paused mRNAs. It has been suggested that a large percentage of transcripts in neuronal processes are stored in these stalled polysomes. Given this, it is predicted that nascent peptides should be abundant in these granules. Nascent peptides can be visualized in real time by the SunTag system. Using this system, we observe nascent peptides in neuronal processes that are resistant to runoff with the initiation inhibitor homoharringtonin (HHT) and to release by puromycin, properties expected from RNA granules consisting of stalled polysomes. In contrast, nascent peptides in nonneuronal cells and neuronal cell bodies were not resistant to HHT or puromycin. Stalled polysomes can also be visualized after runoff with ribopuromycylation and the RNA granules imaged with ribopuromycylation were the same as those with SunTag visualized nascent peptides. Accordingly, the ribopuromycylated puncta in neuronal dendrites were also resistant to puromycin. Thus, the SunTag technique corroborates in situ evidence of stalled polysomes and will allow for the live examination of these translational structures as a mechanism for mRNA transport and regulated protein synthesis.