Functional Interrogation of Brain Microproteins

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The human brain must leverage extraordinary proteomic diversity to receive, process, and execute complex cognitive tasks. To date, while many studies have focused on genomic and transcriptomic regulation in the developing human brain, our understanding of protein translation in this context is limited. Therefore, the long term goal of my research program is to understand how mRNA translation is regulated during neurodevelopment, and how protein evolution contributes to human brain complexity. Recent studies suggest that the proteome is far more diverse than previously appreciated, due in part to the widespread active translation of small open reading frames (sORFs) encoding microproteins =100 amino acids. A range of functions have been ascribed to the small set of microproteins that have been studied in other tissues, including roles as intra- and extracellular second messengers, allosteric regulators of enzymes, and components of macromolecular complexes. The function and physiologic significance of brain-encoded microproteins is unknown, but they represent an exciting and as-yet uncharacterized frontier in neuroscience. To identify candidate microproteins in the human brain, we mapped the translational landscape of the human cortex, from prenatal to adult life, using ribosome profiling. Moreover, we have similarly mapped the `translatome' of human embryonic stem cell (hESC)-derived neurons. From this data, we determined the movement of ribosomes across codons and identified protein-coding ORFs. Our data has revealed the translation of thousands of sORFs, which encode microproteins, from transcripts annotated as non-coding. We found that the majority of microproteins are newly evolved in humans, and many are brain-specific. Interestingly, we found hundreds of microproteins that are functionally related to the RGG domain of intrinsically disordered RNA-binding proteins, suggesting that these microproteins may bind RNA to regulate RNA metabolism or function. This underscores the importance of discerning the protein coding potential of brain-expressed microproteins, which requires further investigation at the level of individual gene candidates. We hypothesize that a subset of microproteins embedded in `non-coding' regions of the genome are critical to neuron survival and function. To test this hypothesis, we propose the following Specific Aims: 1.Characterize the localization and interactome of candidate microproteins in human neurons, with the goal of elucidating biologic function. 2.Perform a CRISPR screen in human neurons to identify high-confidence functional microproteins encoded within annotated non-coding regions. Altogether, these experiments will identify novel regulators of neuron function and survival, and more importantly, provide new insight into a previously under-appreciated class of molecules in the nervous system.