Rapid remodeling of the translatome underlying wound healing and regeneration

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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.