There is emerging evidence from in vitro systems that a remarkable number of mRNAs contain alternative translation initiation sites, further amplifying the number and diversity of protein products that can be generated from a single RNA molecule. This new diversity can be studied in a high throughput and systematic manner in cell culture using newly developed methods for "ribosome footprinting." Here, we propose to bring these new methods into the study of the nervous system using our tools and expertise in capturing ribosomes from genetically defined cell populations in the mouse brain. Thus, Aim 1 is to determine if alternative translation is a common feature of the complex mammalian brain, and how it is regulated across distinct cell types. Furthermore, in yeast and other in vitro systems, alternative translation is regulated by cellular stress and other manipulations.
Our second aim i s to determine whether neuronal activity can regulate alternative translation in vivo, using a newly developed dual-reporter "CHOP-TRAP" mouse that permits parallel optogenetic manipulation and ribosome capture from targeted cell populations, in this case midbrain Dopaminergic neurons. It is our hope that the development of tools to characterize alternative translation in cel populations relevant to reward and addiction will permit future in-depth analysis of this process in CNS disorder.
It has recently been discovered in stem cells that a process called alternative translation, the making of multiple proteins from the same mRNA, occurs surprisingly often. The proposed project tests whether this same process is widespread in the brain, and whether activity in the circuits that regulate addiction and reward can alter this process.