Photoswitchable cell-penetrating PNAs for the manipulation of quiescence during regenerative myogenesis

干细胞小生境的再增殖对于维持许多器官和组织的再生能力至关重要。在骨骼肌中，在所谓的再生肌发生过程中，这部分是通过储备细胞形成过程实现的。在这个过程中，一些已经参与分化的祖细胞（成肌细胞）改变了它们的命运，并通过尚不清楚的机制恢复静止。我们已经确定了nTRIP 6，LIM结构域蛋白TRIP 6的核亚型，作为这一过程的候选调节剂。事实上，这种转录辅助调节因子阻止成肌细胞分化，并富含储备细胞。然而，研究其在体外和体内储备细胞形成中的作用需要工具以高空间和时间精度选择性地阻断其表达或功能，即仅在储备细胞中，并且仅在其命运决定时。我们已经表明，nTRIP 6是通过Trip 6 mRNA中内部AUG的交替翻译产生的，并且靶向该AUG的细胞穿透PNA（PNA-CPP）抑制nTRIP 6的翻译而不影响TRIP 6的翻译。基于我们先前对PNA、CPP和化学光开关的合成和优化的工作，我们的目标是以靶向翻译的光开关PNA-CPP的形式开发新型光化学工具。这些将被应用于研究nTRIP 6在体外肌肉发生和斑马鱼胚胎肌肉再生过程中储备细胞形成中的作用，斑马鱼胚胎是一种特别适合光学方法的动物模型。此外，基于我们将在翻译的光控中获得的经验，我们将开发一种通用的光化学遗传工具，以可逆的方式快速诱导任何感兴趣的蛋白质（POI）的翻译。这个工具，我们称之为SPRINT for Switchable PNAs for the Rapid Induction of Translation，是一个2-组分系统：光开关PNA-CPP（光化学组分）和POI（遗传组分）的表达载体，其中包含抑制POI翻译的顺式作用元件。这些顺式作用元件是PNA的靶标。因此，表达构建体并用光可切换PNA-CPP处理的所选细胞的光暴露将导致POI翻译的快速和可逆诱导。在原理实验的证明中，SPRINT将被应用于在体外和斑马鱼胚胎中成肌细胞恢复静止期间操纵nTRIP 6水平和功能。因此，生物学家和化学家之间的这个合作项目将为储备细胞形成的机制提供新的线索。此外，SPRINT将是对目前以非常高的空间和时间精度操纵生物系统的工具箱的重要补充。

The repopulation of stem cell niches is essential to maintain the regeneration capacity of many organs and tissues. In skeletal muscle, during the so-called regenerative myogenesis, this is achieved in part by the process of reserve cells formation. In this process, some progenitor cells (myoblasts) that were already engaged in differentiation change their fate and return to quiescence by poorly understood mechanisms. We have identified nTRIP6, the nuclear isoform of the LIM domain protein TRIP6, as a candidate regulator of this process. Indeed, this transcriptional co-regulator prevents myoblast differentiation and is enriched in reserve cells. However, the study of its role in reserve cell formation in vitro and in vivo requires tools to selectively block its expression or function with high spatial and temporal precision, i.e. only in reserve cells, and only at the time of their fate decision. We have shown that nTRIP6 is generated by alternative translation at an internal AUG in Trip6 mRNA, and that a cell-penetrating PNA (PNA-CPP) targeting this AUG inhibits the translation of nTRIP6 without affecting that of TRIP6. Based on our previous work on the synthesis and optimisation of PNAs, CPPs and chemical photoswitches, our aim I to develop novel optochemical tools in the form of photoswitchable PNA-CPPs that target translation. These will be applied to investigate the role of nTRIP6 in the formation of reserve cells during myogenesis in vitro and muscle regeneration in zebrafish embryos, an animal model particularly amenable to optical methods. Furthermore, based on the experience we will gain in the photocontrol of translation, we will develop a generic optochemical genetic tool to rapidly induce the translation of any protein of interest (POI) in a reversible manner. This tool, which we call SPRINT for Switchable PNAs for the Rapid INduction of Translation, is a 2-component system: a photoswitchable PNA-CPP (optochemical component) and an expression vector for the POI (genetic component), which contains cis-acting elements that repress the translation of the POI. These cis-acting elements are the target of the PNA. Thus, light exposure of selected cells expressing the construct and treated with the photoswitchable PNA-CPP will lead to the rapid and reversible induction of the POI translation. In proof of principle experiments, SPRINT will be applied to the manipulation of nTRIP6 levels and function during myoblast return to quiescence in vitro and in the zebrafish embryo. Thus, this collaborative project between a biologist and a chemist will shed new light onto the mechanism of reserve cell formation. Furthermore, SPRINT will represent an important addition to the current toolbox for the manipulation of biological systems with very high spatial and temporal precision.