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

项目摘要 人类的发展依赖于高度协调的细胞分裂、信号传递、迁移和分化。 协调这些关键过程的蛋白质效应器的任何扰动都可能导致人类出生缺陷 和/或疾病1-3。虽然我们的经典目录包含大约20,000种蛋白质，但基于组学的证据 技术已经引起了RNA生物学的快速范式转变4-12。值得注意的是，被定义为非 事实上，编码产生短蛋白质(GB 100个氨基酸)，称为微蛋白，调节不同的过程。 40，包括心脏功能26，免疫22，27，41和细胞生长29，30。例如，一种名为apela的微蛋白 维持人类胚胎干细胞的多能性20，对斑马鱼心脏发育至关重要。 然而，脊椎动物发育过程中的额外微蛋白功能(S)在很大程度上仍不清楚。 斑马鱼是研究脊椎动物基因功能的优秀模型。他们的遗传可塑性 再加上外部的同步发展，非常适合于发展分析。此外，还有数百人 利用核糖体图谱，在斑马鱼发育过程中鉴定了大量的微蛋白。 光谱分析和守恒分析16，42。值得注意的是，Apela是这400种蛋白质中唯一一种 目前的特点是。进一步研究微蛋白的一个关键障碍是大多数信使RNA (信使核糖核酸)在早期发育是母体提供的，可以掩盖目标基因的影响 颠覆。我们的新型CRISPR/Cas13d系统43、44克服了这一障碍，因为它主动降解了它的目标 因此，能够选择性地敲除斑马鱼中母体提供的mRNAs。我的初选 CRISPR/Cas13d的实验表明，敲除一种微蛋白mRNA可以抑制受精卵 基因组激活并扰乱后部模式。 这项研究将结合CRISPR/Cas13d和基于组学的技术来询问微蛋白质 在斑马鱼发育过程中发挥作用。AIM 1将利用CRISPR/Cas13d来阐明重要的微蛋白 用于早期开发。然后，目标2将确定依赖于发育的细胞和分子过程 微蛋白。总而言之，这些目标将定义和表征一组参与 脊椎动物的发育。实验方法将培养我在生物信息学、分子遗传学、 发育生物学和蛋白质生物化学。对斑马鱼发育至关重要的微蛋白将是 为通过比较分析扩展人类蛋白质的目录提供了信息。此外，微蛋白 在发育过程中的功能代表着人类未知的治疗和/或诊断机会 先天缺陷和/或人类发育疾病。

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.