Opening small packages: unraveling roles for microproteins during early vertebrate development

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Project Summary Human development relies on highly coordinated cell division, signaling, migration, and differentiation. Any perturbations of the protein effectors that orchestrate these critical processes can lead to human birth defects and/or diseases1-3. While our classical catalog contains around 20,000 proteins, evidence from ‘omics-based techniques has generated a rapid paradigm shift in RNA biology4-12. Notably, RNA sequences defined as non- coding in fact produce short proteins (£ 100 amino acids) called microproteins that modulate diverse processes13- 40 including heart function26, immunity22,27,41, and cell growth29,30. For example, a microprotein called APELA maintains pluripotency in human embryonic stem cells20 and is critical for zebrafish heart development16,19. However, additional microprotein function(s) during vertebrate development remain largely unknown. Zebrafish is an outstanding model for interrogating vertebrate gene function. Their genetic tractability coupled with external, synchronous development is well-suited for developmental analyses. Further, hundreds of microproteins have been identified across zebrafish development using ribosome profiling, mass- spectrometry, and conservation analyses16,42. Remarkably, APELA is the only microprotein out of these 400 that is currently characterized. A key barrier to further microprotein investigation is that a majority of messenger RNAs (mRNA) during early development are maternally provided and can mask the effects of a targeted gene disruption. Our novel CRISPR/Cas13d system43,44 overcomes this barrier because it actively degrades its target mRNA and therefore enables selective knockdown of maternally provided mRNAs in zebrafish. My preliminary experiments with CRISPR/Cas13d have revealed that knockdown of one microprotein mRNA inhibits zygotic genome activation and disrupts posterior patterning. This study will combine CRISPR/Cas13d and ‘omics-based techniques to interrogate microprotein function during zebrafish development. Aim 1 will leverage CRISPR/Cas13d to elucidate microproteins important for early development. Then, Aim 2 will determine the cell and molecular processes that rely on developmental microproteins. Together, these aims will define and characterize a population of microproteins involved in vertebrate development. Experimental approaches will develop my skills in bioinformatics, molecular genetics, developmental biology, and protein biochemistry. Microproteins critical for zebrafish development will be informative for expanding the catalog of human proteins through comparative analyses. Further, microproteins with functions during development represent uncharted therapeutic and/or diagnostic opportunities for human birth defects and/or human diseases with developmental origins.