Protection of Drosophila development through the regulation of alternative splicing in the germline

该项目旨在了解果蝇正常发育的生殖系中调节选择性剪接的机制。研究的主要重点将是了解pirna如何调节果蝇种系中p元素DNA转座子的剪接，从而防止由p元素转座子引起的一系列发育缺陷（称为杂交发育不良），包括不育和高水平突变。母系遗传的pirna在种系中的存在导致含有过早终止密码子的p元件内含子的保留，阻止编码转座酶的完全翻译并抑制该元件的转座。在经典的piRNA通路中，许多已知的调节染色质状态变化的因子与抑制内含子的剪接有关，通过先前的工作显示这些因子的敲除会导致剪接抑制。此外，这种抑制与整个元件中H3K9三甲基化的整体增加有关。在研究这个选择性剪接调节的例子中，目的是了解染色质如何参与调节剪接的机制以及需要哪些因素。了解这一点的潜在实验可能包括dcas9介导的染色质修饰蛋白募集到元件以评估剪接效应，在存在或不存在这种调节的情况下p元素相关因子的质谱分析，以及通过GRO-Seq和使用改变速度的聚合酶突变体的分析来评估RNA聚合酶II活性的变化。以前的工作已经将其他基因的染色质变化与选择性剪接联系起来，所以如果在这里起作用的机制可以被阐明，进一步的工作可以确定它们是否适用于其他选择性剪接调节的例子。通过对内源性发育基因剪接调节的研究，也有可能扩大这项工作，以便更广泛地了解果蝇种系中如何调节选择性剪接以防止发育缺陷。

PhD project strategic theme: Understanding the rules of lifeThis project aims to understand the mechanisms regulating alternative splicing in the germline that allow Drosophila development to occur normally. The primary focus will be on understanding the mechanism through which piRNAs regulate splicing of the P-element DNA transposon in the Drosophila germline, preventing a series of developmental defects (called hybrid dysgenesis) resulting from P-element transposition, which include sterility and high levels of mutation. The presence of maternally inherited piRNAs in the germline leads to retention of a P-element intron containing a premature stop codon, preventing full translation of the encoded transposase and suppressing transposition of the element. A number of factors that are known to modulate changes in chromatin states in classical piRNA pathways are implicated in repressing splicing of this intron, through previous work showing splicing derepression upon knockdown of these factors. Additionally, this repression is associated with a global increase in H3K9 trimethylation across the element. In studying this example of alternative splicing regulation, the aim is to understand the mechanism of how chromatin is involved in regulating splicing and which factors are required. Potential experiments to understand this may include dCas9-mediated recruitment of chromatin-modifying proteins to the element to assess splicing effects, mass spectrometry of P-element-associating factors in the presence/absence of this regulation and assessing changes in RNA polymerase II activity through GRO-Seq and assays using polymerase mutants with altered speed. Previous work has linked chromatin changes with alternative splicing in the cases of other genes, so if the mechanisms at play here can be elucidated, further work could determine whether they apply to other examples of alternative splicing regulation. There is also the potential to expand this work in order to more broadly understand how alternative splicing is regulated in the Drosophila germline to prevent developmental defects, through the study of splicing regulation in endogenous developmental genes.