Darshan’s 2018 pre-publication profiles the process of mRNA-to-protein translation in neurons and astrocytes in both cell culture and the living mouse brain. Ribosome footprinting (RF) is a modified version of RNA-sequencing that maps where on mRNAs ribosomes are bound (i.e. translating or scanning for start sites); this technique forms the basis of the results presented here. A key innovation in acquiring some of the cutting edge in vivo data was using TRAP to capture ribosomes and their bound mRNAs from single CNS cell types, and performing RF on the isolated TRAP ribosome-mRNAs. As a result, Sapkota, et. al present independent, in vivo snapshots of translation occurring in neurons and astrocytes. These data reveal cell-type specific translational events, including alternative initiation of translation and alternative stop codon usage in SNAP25-positive neurons and Aldh1l1-positive astrocytes from the mouse brain.
•Translation initiation sites. These come from HHT treatment of neuron-astrocyte co-culture in vitro. (HHT arrests translation-initiation but allows translating ribosomes to run off the transcripts, thus enabling ribosome footprinting specifically of translation initiation).
•Full-transcript ribosome footprinting of neuron-astrocyte co-cultures. These cultures were not treated with HHT–only cycloheximide (CHX), which arrests ribosomes wherever they are on an mRNA. This data is publicly available through GEO.
•Cell-type specific ribosome footprinting of neurons or astrocytes from mouse brain. This data, which leveraged TRAP as a means of acquiring RF data, is on GEO with an anticipated release date of January 14, 2019.
Please direct any science questions to Darshan by e-mail at: dsapkota [at] wustl DOT edu. Any technical questions (i.e., can’t access the files), contact Bernie by e-mail at berniejmulvey [at] gmail DOT com. Have fun!