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

打开小包装：揭示微生物蛋白在早期脊椎动物发育过程中的作用

• 批准号: 10678492
• 负责人: Anthony J Treichel
• 金额: $ 3.17万
• 依托单位: STOWERS INSTITUTE FOR MEDICAL RESEARCH
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10678492
• 关键词: Affect; Amino Acids; Base Sequence; Biochemical; Bioinformatics; Biology; Catalogs; Cell division; Cell physiology; Cells; Classification; Clustered Regularly Interspaced Short Palindromic Repeats; Computational Biology; Congenital Abnormality; Coupled; Data; Data Set; Development; Developmental Biology; Diagnostic; Disease; Down-Regulation; Embryo; Embryonic Development; Epitopes; Etiology; Exclusion; Exhibits; Gene Expression; Genes; Genetic; Genetic Transcription; Genome; Germ Layers; Goals; Guide RNA; Hour; Human; Human Development; Image; Immunity; Immunofluorescence Immunologic; Investigation; Link; Masks; Mass Spectrum Analysis; Maternal Messenger RNA; Messenger RNA; Microscopy; Modeling; Molecular; Molecular Biology; Molecular Genetics; Open Reading Frames; Pathway interactions; Pattern; Phenotype; Play; Population; Positioning Attribute; Process; Protein Biochemistry; Proteins; Proteomics; Publishing; Quality of life; RNA; RNA Interference; RNA Sequences; Role; Signal Transduction; Specific qualifier value; System; Techniques; Testing; Therapeutic; Training; Translating; Translations; Untranslated RNA; Untranslated Regions; Variant; Zebrafish; cardiogenesis; cell growth; cell motility; comparative; developmental disease; experimental study; gastrulation; gene function; grasp; heart function; human disease; human embryonic stem cell; image guided; imaging approach; improved; in vivo; innovation; insight; knock-down; migration; mutant; novel; offspring; pluripotency; protein function; ribosome profiling; skills; spatiotemporal; transcription factor; transcriptome sequencing; zebrafish development; zygote

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.

项目概要 人类发育依赖于高度协调的细胞分裂、信号传导、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，但来自“基于组学”的证据 技术使 RNA 生物学发生了快速的范式转变4-12。值得注意的是，RNA 序列定义为非 事实上，编码会产生短蛋白质（100 英镑氨基酸），称为微生物蛋白质，可调节不同的过程13- 40 包括心脏功能26、免疫力22,27,41 和细胞生长29,30。例如，一种名为 APELA 的微生物蛋白 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16,19。 然而，脊椎动物发育过程中额外的微生物蛋白功能仍然很大程度上未知。 斑马鱼是研究脊椎动物基因功能的杰出模型。他们的遗传易处理性 与外部同步开发相结合非常适合开发分析。此外，数百 使用核糖体分析、质量分析在斑马鱼发育过程中鉴定了微生物蛋白 光谱测定和守恒分析16,42。值得注意的是，APELA 是这 400 种微生物蛋白质中唯一一种 目前已被表征。进一步微生物蛋白研究的一个关键障碍是大多数信使 RNA (mRNA) 在早期发育过程中由母体提供，可以掩盖目标基因的影响 扰乱。我们的新型 CRISPR/Cas13d 系统43,44 克服了这一障碍，因为它主动降解其靶标 因此能够选择性地敲低斑马鱼中母体提供的 mRNA。我的初步 CRISPR/Cas13d 实验表明，敲低一种微生物蛋白 mRNA 会抑制合子 基因组激活并破坏后模式。 这项研究将结合 CRISPR/Cas13d 和基于组学的技术来探究微生物蛋白 斑马鱼发育过程中的功能。目标 1 将利用 CRISPR/Cas13d 阐明重要的微生物蛋白 以利于早期发展。然后，目标 2 将确定依赖于发育的细胞和分子过程。 微生物蛋白质。这些目标共同将定义和表征参与以下过程的微生物蛋白群体： 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生物蛋白将是 通过比较分析为扩大人类蛋白质目录提供信息。此外，微生物蛋白 在开发过程中具有的功能代表了人类未知的治疗和/或诊断机会 出生缺陷和/或发育起源的人类疾病。