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

博士项目战略主题：了解生命的规则这个项目的目的是了解调节生殖细胞中选择性剪接的机制，使果蝇的发育正常发生。主要重点将是了解piRNA调节果蝇生殖系中P元件DNA转座子剪接的机制，防止由P元件转座导致的一系列发育缺陷（称为杂种发育不良），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提前终止密码子的P元件内含子的保留，从而阻止编码的转座酶的完全翻译并抑制元件的转座。已知在经典皮尔纳途径中调节染色质状态变化的许多因子涉及抑制该内含子的剪接，通过先前的工作显示在敲低这些因子后的剪接去抑制。此外，这种抑制与整个元素中H3 K9三甲基化的全球增加有关。在研究这个选择性剪接调控的例子时，目的是了解染色质如何参与调控剪接的机制以及需要哪些因子。理解这一点的潜在实验可能包括dCas 9介导的染色质修饰蛋白向元件的募集以评估剪接效应，在存在/不存在这种调节的情况下对P元件相关因子进行质谱分析，以及通过GRO-Seq和使用具有改变的速度的聚合酶突变体的测定来评估RNA聚合酶II活性的变化。先前的工作已经将染色质变化与其他基因的选择性剪接联系起来，因此如果这里的机制能够得到阐明，进一步的工作可以确定它们是否适用于选择性剪接调控的其他例子。也有可能扩大这项工作，以更广泛地了解选择性剪接是如何调节果蝇种系，以防止发育缺陷，通过内源性发育基因的剪接调控的研究。

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.