Targeted mRNA degradation in Drosophila spermatogenesis

干细胞在再生医学中具有巨大的潜力，可以替代有缺陷的组织。因此，它们为损伤和退行性疾病（如阿尔茨海默氏症和杜氏肌营养不良症）提供了潜在的治疗方法。在睾丸中，需要干细胞来维持精子的供应，这样男性才有可能在一生的大部分时间里生育后代。近年来，对基因的理解有了快速的进展，这些基因需要被打开才能产生自我更新的干细胞，这些干细胞能够发育成各种组织，如皮肤或神经组织。然而，现在我们知道，确保某些基因被关闭以防止干细胞自发地分化成特定的细胞类型或发展成癌细胞也是至关重要的。我们最近在果蝇的睾丸细胞中发现了一种名为Pacman的酶，它参与了信使RNA分子的破坏，是干细胞功能所必需的。信使rna是指示细胞制造特定蛋白质的分子。通过比较突变果蝇和正常果蝇，我们发现吃豆人突变果蝇的睾丸干细胞较少，导致精子和后代较少。使用各种遗传和分子技术，我们发现吃豆人可能会破坏特定的rna，否则会阻止干细胞分裂或细胞死亡。这很有趣，因为它表明这些mrna必须以某种方式被“标记”，这样吃豆人及其伙伴才能识别、“追捕”并摧毁这些特定的rna。这个项目的具体目标是，从分子的角度来理解，吃豆人及其合作伙伴是如何识别和破坏其正确的目标rna的，以及这是如何控制的。吃豆人选择性破坏目标rna的能力并不是它本身固有的，因为试管中分离的吃豆人蛋白不能区分不同的rna。我们知道不同的rna在不同的组织中被选择那么吃豆人和它的伙伴是如何做到的呢？我们的假设是，一个特定的蛋白质（或一个微小的调节RNA）与一个特定的特征或目标RNA结合，并“标记”它的破坏。这个标签随后被吃豆人及其伙伴蛋白识别。我们认为，吃豆人是一种大蛋白，可以作为支架在其上组装其他蛋白质。当正确的伙伴以正确的三维形状组装起来时，一种斩首酶就会剪掉RNA的末端，剩下的部分就会被迅速咀嚼掉。在这个项目中，我们的目标是找出这种破坏途径的细节，包括标记目标的方式。这项工作将增加我们对基因在细胞环境下被特异性关闭的方式的一般理解。由于已知吃豆人在其他重要的细胞事件中也很重要，如伤口愈合和细胞片的迁移，这项工作也可能揭示这些过程的分子机制。虽然这个项目将在果蝇身上进行，但它也与人类的干细胞功能有关，因为这两种生物的细胞过程惊人地相似。此外，吃豆人酶在果蝇和人类之间非常相似，因此我们在这个项目中获得的见解可能有助于我们改善生育治疗，也有助于我们理解干细胞在生物体的生命周期中保持几乎不朽的方式。因此，该项目将为特定rna靶向特定细胞类型的方式提供有价值的见解，可用于开发新疗法。

Stem cells have a vast potential in regenerative medicine for the replacement of defective tissue. They therefore offer a potential cure for injuries and also for degenerative diseases such as Alzheimers and Duchenne muscular dystrophy. In the testis, stem cells are required to maintain the supply of sperm, so that the male has potential to produce offspring for much of his life. In recent years, there have been rapid advances in the understanding of the genes which are required to be switched ON to produce self-renewing stem cells that are capable of developing into a wide range of tissues e.g. skin or nervous tissue. However, it is now known that it is also crucially important to make sure that certain genes are switched OFF to prevent stem cells from spontaneously differentiating into particular cell types or developing into cancer cells. We have recently discovered that an enzyme named Pacman, which is involved in the destruction of messenger RNA molecules, is necessary for stem cell function in testis cells of the fruit fly Drosophila. Messenger RNAs are the molecules which instruct the cell to make particular proteins. By comparing mutant flies with normal flies, we have found out that there are fewer testis stem cells in the pacman mutant leading to fewer sperm and offspring. Using various genetic and molecular techniques we have found out that Pacman is likely to destroy particular RNAs that would otherwise prevent stem cell division or cell death. This is interesting as it shows that these mRNAs must somehow be 'tagged' allowing Pacman and its partners to identify, 'hunt down' and destroy these particular RNAs. The specific aim of this project is to understand, in molecular terms, exactly how Pacman and its partners can identify and destroy its correct target RNAs and how this is controlled. The ability of Pacman to selectively destroy target RNAs is not intrinsic to itself as isolated Pacman protein in the test tube cannot distinguish between different RNAs. We know that different RNAs are selected in different tissues so how does Pacman and its partners do this? Our hypothesis is that a particular protein (or a tiny regulatory RNA) binds to a specific feature or the target RNA and 'tags' it for destruction. This tag is then recognised by Pacman and its partner proteins. We propose that Pacman, which is a large protein, can act as a scaffold to assemble other proteins upon it. When the correct partners are assembled, in the correct 3-dimensional shape, then a decapitation enzyme snips off the end of the RNA and the rest is rapidly chewed up. In this project, we aim to find out the details of this destruction pathway, including the ways that the target is tagged. This work will increase our general understanding of the ways that genes are specifically switched off in response to their cellular context. Since Pacman is known to be important in other important cellular events such as wound healing and migration of cell sheets, this work may also shed light on the molecular mechanisms of these processes. Although this project will be carried out using the fruit fly Drosophila, it also has relevance for stem cell function in humans as the cellular processes are surprisingly similar in both organisms. In addition, the Pacman enzyme is extremely similar between flies and humans therefore the insights we gain during this project may help us to improve treatment for fertility and also help us to understand the ways that stem cells remain virtually immortal for the lifetime of the organism. This project will therefore provide valuable insights into the ways in which specific RNAs are targeted in a particular cell type which can be used in the development of new therapeutics.