Addressing Protein Synthesis Regulation within Small Numbers of Discrete Neurons

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Project Summary/Abstract Transcription dominates the gene expression landscape of circadian rhythms and a number of neuroscience areas. Yet post-transcriptional regulation, including translational regulation and the role of RNA binding proteins (RBPs), has become increasingly recognized as important in recent years. Moreover, understanding the roles of RBPs in diverse cell types and diseases and ultimately therapeutic intervention requires identifying the RNA targets of RBPs. The ribosome can be considered a RBP, so this focus on post-transcriptional regulation and RBP targets includes identifying ribosome-associated transcripts, namely RNAs that change their translational status under defined circumstances. RBP identification is particularly challenging from small numbers of cells, e.g., cancer stem cells within a large, heterogeneous solid tumor or discrete neuronal subtypes. These settings preclude traditional biochemistry and therefore require new approaches. Two new methods recently appeared, TRIBE and STAMP, which exploit the RNA editing enzymes ADAR and APOBEC, respectively. Their ribosome versions, Ribo-STAMP and Ribo-TRIBE, are even more recent and fuse a ribosomal protein to the editing enzymes. This is so that the enzyme will be near RNAs that are being translated and will “mark” them by changing their sequence. These edits are identified by mRNA sequencing and straightforward computational methods, even from single cells. We propose to compare Ribo-STAMP and Ribo-TRIBE side-by-side in mammalian cell culture systems, to assess their efficacy and to determine the optimal configuration of ribosome-editing enzyme fusions. We also propose to develop Ribo-TRIBE for use in Drosophila; we recently discovered that STAMP does not work in this organism, which limits us to TRIBE. We will then extend these methods to the more biological context of neurons, from Drosophila as well as from mouse brains. To complement these efforts, we will develop an extension of the TRIBE/STAMP theme called Nanobody-editing. It consists of fusing the editing enzyme, ADAR or APOBEC, to a GFP-nanobody, which will then deliver the editing enzyme to any GFP-tagged RNA binding protein or GFP-tagged ribosome. The chimeric, recombinant protein will be used in vitro as a recombinant protein or expressed in vivo. Nanobody- editing will facilitate identifying RBP and ribosome targets, because already existing GFP-tagged RBP or ribosomes can serve as substrates. Moreover, in vitro editing will in many cases obviate the need to generate new transgenes and transgenic animals. Lastly, TRIBE and as well as Ribo-TRIBE will be used to characterize translational regulation “around the clock” within a few key Drosophila circadian neurons. These efforts will deepen our understanding of circadian gene expression regulation as well as provide the community with new tools with which to study translation in important but challenging biological systems.