Rapid remodeling of the translatome underlying wound healing and regeneration

伤口愈合和再生中翻译组的快速重塑

• 批准号: 10445695
• 负责人: Maria Barna
• 金额: $ 34.2万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10445695
• 关键词: Adult; Ambystoma; Ambystoma mexicanum; Amputation; Animals; Binding Sites; Brain; Cell Fate Control; Cell Proliferation; Cell Survival; Cells; Complex; Cytoplasmic Granules; Development; Digit structure; Disease; Evolution; Exhibits; FRAP1 gene; Foundations; Genetic Transcription; Genetic Translation; Goals; Growth; Guanosine Triphosphate Phosphohydrolases; Health; Heart; Human; Injury; Knock-in; Light; Limb structure; Lysosomes; Mammals; Membrane; Messenger RNA; Methods; Molecular; Mus; Natural regeneration; Organ; Organism; Pathway interactions; Phosphotransferases; Polyribosomes; Production; Protein Biosynthesis; Proteins; RNA, Messenger, Stored; Regulation; Resolution; Rest; Ribosomal Proteins; Ribosomes; Role; Salamander; Science; Signal Transduction; Site; Spinal Cord; Stress; Stress Response Signaling; Structure; Technology; Testing; Time; Tissues; Transcript; Translating; Translational Activation; Translational Regulation; Translational Research; Translations; Vertebrates; base; cell fate specification; genome-wide; healing; human tissue; limb amputation; limb regeneration; mouse model; novel; polysome profiling; protein activation; recruit; regeneration potential; regenerative; repaired; response; restoration; ribosome profiling; selective expression; severe injury; tissue regeneration; tool; transcriptome; transcriptome sequencing; translational impact; translatome; wound healing

The biggest biomedical challenge of this century is the restoration of diseased organs and tissues. Unlike humans, salamanders have the extraordinary ability to rapidly regenerate organs, including limbs, spinal cords, hearts and brains. Our goal is to discover how these animals rebuild functional adult tissues in a matter of weeks. From development through degeneration – the health and function of our organs depends on production of appropriate tissue-specific proteins. Yet, our current understanding of regeneration is largely based on studies of mRNA and not on direct assessment of proteins that are ultimately required for repair. This is in part due to technical limitations – microarray and RNA-Seq technologies revolutionized our understanding of transcription- but until recently we lacked the tools to study translation of mRNA into protein at the same scale and resolution. The Mexican axolotl is famous for its lifelong “youthfulness”. Axolotls share with other salamanders the surprising and incompletely understood ability to regrow entire limbs after amputation. By combining cutting-edge methods in translation research, we were able to demonstrate that, unlike in mammals, severe injury in the axolotl surprisingly results in rapid activation of protein synthesis at a time when there is little cellular proliferation. This unusual molecular response is a feature specific to regenerative vertebrates and relies on activation of the mammalian target of rapamycin (mTOR) pathway. Moreover, we find that remarkably fewer than 20% of all axolotl mRNAs are translated at any given time, the remainder exist in a ‘free’ state outside the translation machinery. We will test the hypothesis that the ‘free’ transcripts in the axolotl may be spatially organized into membrane-less compartments comprised of functionally-related and translationally co-regulated mRNAs and that transcripts critical for cell survival and cell fate specification shuttle between these compartments and the ribosome to facilitate wound healing and regeneration. We have further identified that control of protein synthesis at the time of regeneration is highly dependent on the ability of the Axolotl to surpass a stress activating signal and instead promote activation of the mTOR pathway. We will test the hypothesis that the structural/sequence specific differences in Axolotl mTOR components can shed light on functional differences in upstream regulation of protein synthesis between species and the remarkably ability to repurpose a ‘stress-response’ signal to a ‘growth and regeneration’ signal. These findings suggest the possibility that poor healing in mammals may be due to a distinct cellular signaling response at the site of injury rather than to an inherent lack of regenerative potential. Lastly, we have found that amputation of the limb in the axolotl triggers selective translation of some ribosomal proteins but not others, coincident with the “burst” in protein synthesis. We will therefore test the bold hypothesis that axolotls may assemble distinct subsets of specialized ribosomes to facilitate selective expression of transcripts critical for wound healing and regeneration. Together, this proposal seeks to provide a novel mechanistic understanding as to why some species can regenerate while others cannot.

本世纪最大的生物医学挑战是修复患病的器官和组织。不像 与人类相比，蝾螈具有快速再生器官的非凡能力，包括四肢，脊髓， 心脏和大脑我们的目标是发现这些动物如何在几周内重建功能性成年组织。 从发育到退化-我们器官的健康和功能取决于生产 合适的组织特异性蛋白质。然而，我们目前对再生的理解主要是基于研究 而不是直接评估最终修复所需的蛋白质。这部分是由于 技术限制-微阵列和RNA-Seq技术彻底改变了我们对转录的理解- 但直到最近，我们还缺乏以同样的尺度和分辨率研究mRNA翻译成蛋白质的工具。 墨西哥美西蝾螈以其终生的“青春”而闻名。蝾螈和其他蝾螈一样 以及截肢后再生整个肢体的能力尚不完全清楚。通过结合尖端的方法 在翻译研究中，我们能够证明，与哺乳动物不同， 令人惊讶的是，在几乎没有细胞增殖的情况下迅速激活蛋白质合成。这 不寻常的分子反应是再生脊椎动物特有的特征， 哺乳动物雷帕霉素靶蛋白（mTOR）途径。此外，我们发现， 蝾螈mRNA在任何给定的时间翻译，其余的存在于翻译外的“自由”状态 机械.我们将检验这一假设，即蝾螈中的“自由”转录本可能在空间上组织成 由功能相关和功能共调节的mRNA组成的无膜区室， 对于细胞存活和细胞命运规范至关重要的转录物在这些区室之间穿梭， 核糖体以促进伤口愈合和再生。我们进一步证实了蛋白质合成的控制 在再生的时候是高度依赖于能力的蝾螈超越应力激活信号 而是促进mTOR通路的激活。我们将检验结构/序列 蝾螈mTOR组分的特定差异可以揭示上游调控的功能差异 物种之间的蛋白质合成，以及将“压力反应”信号重新用于 “生长和再生”信号。这些发现表明，哺乳动物的不良愈合可能是 这是由于在损伤部位的不同细胞信号传导反应，而不是由于固有的再生能力缺乏， 潜力最后，我们发现，美西螈的截肢触发了一些选择性翻译， 核糖体蛋白质，而不是其他蛋白质，与蛋白质合成的“爆发”相一致。因此，我们将测试大胆的 假设蝾螈可能组装特殊核糖体的不同子集以促进选择性表达 对伤口愈合和再生至关重要的转录物。总之，该提案旨在提供一种新颖的 为什么有些物种可以再生而另一些不能。