Evolutionary and functional analysis of non-canonical ribosomal protein paralogues in the Drosophila germline

果蝇种系中非经典核糖体蛋白旁系同源物的进化和功能分析

• 批准号: 2499297
• 金额: --
• 依托单位: University of Cambridge
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2019
• 资助国家: 英国
• 起止时间: 2019 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2499297
• 关键词: Evolutionary; functional; analysis; non; canonical

The central dogma of biology states that "DNA makes RNA makes protein". There has been much research into the regulation of gene expression at the transcriptional level and it is known that the action of RNA polymerase II is highly regulated, however, there has been far less research into the regulation of gene expression at the translational level. Protein synthesis is essential for cell proliferation so, unsurprisingly, the production of sufficient functional ribosomes is hugely important for development, and a variety of human diseases known as ribosomopathies result from mutations in ribosomal proteins or biogenesis factors. The current explanation for the pathogenesis of both of these diseases is that reduced expression levels of specific ribosomal proteins or biogenesis factors leads to fewer ribosomes being assembled, resulting in a lower rate of translation, which is insufficient for cell proliferation. However, this cannot explain the difference in phenotypes caused by mutations in different ribosomal proteins. Accumulating evidence indicates that intrinsic regulation of the translational apparatus provides an additional layer of gene expression control. Such regulation can be achieved in a variety of ways, including the incorporation of ribosomal protein paralogues, posttranscriptional modifications of ribosomal RNAs, and the activity of ribosome-associated factors. An increasing number of examples of "specialised ribosomes" have been described from plants to humans, however, the extent and function of ribosomal heterogeneity is still poorly understood, as research is made difficult due to the lethality of most ribosomal protein knock-downs. The germline does not impose these constraints and is therefore an ideal tissue for research.During an in vivo RNAi screen in Drosophila germline stem cells (GSCs), many factors were identified as being required for specific aspects of GSC development, including ribosome biogenesis factors and ribosomal protein paralogues. The goal of this project is to focus on the role of ribosomal protein paralogues in the germline. Many paralogues were shown to be enriched in undifferentiated germ cells (including RpL22-like, RpS19b, RpS5b, RpL37b, and RpS10a). In addition, germline knockdown of individual ribosomal subunits generally leads to germ cell loss, however, unpublished results indicate that germline knockdown of some individual ribosome protein paralogues (such as RpL22 and RpL22-like) does not affect cell survival, but instead blocks stem cell differentiation. These results support the idea that heterogeneity in ribosome composition underlies key aspects of fate transitions. As germline knockouts are not lethal, I should be able to generate knockout mutants of most ribosomal protein paralogues using CRISPR/Cas9 technology. I will characterise the developmental defects of these mutants through a combination of genetics and immunofluorescent imaging, to understand whether these paralogues are required for fertility, viability or stem cell maintenance. In parallel, genomic-tagged loci will be used to provide a detailed developmental understanding of the expression and localisation of different paralogues. Finally, I will investigate whether these ribosomal protein paralogues are assembled into ribosomes. It is also important to consider possible non-ribosomal roles - some ribosomal proteins are already known to have non-ribosomal roles, for example RpL22 interacts with histone H1 and is associated with the formation of heterochromatin. If the paralogues are indeed assembled into ribosomes, I will investigate whether the translatomes of these ribosomes differ from those of ribosomes not containing the paralogues. Based on this analysis, I aim to uncover the molecular difference between translational machineries, and propose a consolidated model of how heterogeneity in ribosome composition modulates fate transitions.

生物学的中心法则是“DNA制造RNA制造蛋白质”。对转录水平上的基因表达调控已经进行了大量研究，并且已知RNA聚合酶II的作用受到高度调控，然而，对翻译水平上的基因表达调控的研究少得多。蛋白质合成对于细胞增殖是必不可少的，因此，毫不奇怪，产生足够的功能性核糖体对于发育非常重要，并且各种称为核糖体病的人类疾病都是由核糖体蛋白或生物发生因子的突变引起的。目前对这两种疾病发病机制的解释是，特定核糖体蛋白或生物合成因子的表达水平降低导致组装的核糖体减少，导致翻译速率降低，这不足以促进细胞增殖。然而，这不能解释不同核糖体蛋白突变引起的表型差异。越来越多的证据表明，翻译装置的内在调节提供了另一层基因表达控制。这种调节可以通过多种方式实现，包括掺入核糖体蛋白旁系同源物、核糖体RNA的转录后修饰和核糖体相关因子的活性。从植物到人类，描述了越来越多的“特化核糖体”的例子，然而，核糖体异质性的程度和功能仍然知之甚少，因为由于大多数核糖体蛋白质敲低的致命性，研究变得困难。在果蝇生殖系干细胞（GSC）的体内RNAi筛选中，许多因子被鉴定为GSC发育的特定方面所需，包括核糖体生物发生因子和核糖体蛋白旁系同源物。这个项目的目标是关注核糖体蛋白旁系同源物在生殖系中的作用。许多旁系同源物显示富集在未分化的生殖细胞中（包括RpL 22样、RpS 19 b、RpS 5 b、RpL 37 b和RpS 10 a）。此外，单个核糖体亚基的种系敲低通常导致生殖细胞损失，然而，未发表的结果表明，一些单个核糖体蛋白旁系同源物（如RpL 22和RpL 22样）的种系敲低不影响细胞存活，而是阻断干细胞分化。这些结果支持了这样的想法，即核糖体组成的异质性是命运转变的关键方面。由于种系敲除不是致命的，我应该能够使用CRISPR/Cas9技术产生大多数核糖体蛋白旁系同源物的敲除突变体。我将通过遗传学和免疫荧光成像的结合来检测这些突变体的发育缺陷，以了解这些旁系同源物是否是生育力、生存力或干细胞维持所必需的。与此同时，基因组标记的位点将被用来提供一个详细的发展理解的表达和定位不同的旁系同源物。最后，我将研究这些核糖体蛋白旁系同源物是否组装到核糖体中。考虑可能的非核糖体作用也很重要-一些核糖体蛋白已经知道具有非核糖体作用，例如RpL 22与组蛋白H1相互作用并与异染色质的形成有关。如果旁系同源物确实组装成核糖体，我将研究这些核糖体的翻译组是否与不含旁系同源物的核糖体的翻译组不同。基于这种分析，我的目标是揭示翻译机制之间的分子差异，并提出一个统一的模型，如何在核糖体组成的异质性调节命运的转变。