Isoform expression and post-transcriptional regulation of centrosomal plp mRNA

项目摘要 中心体失调与发育障碍有关，如小头畸形、纤毛病变和 心血管疾病尽管它们对人类健康至关重要， 关于编码核心中心体成分的基因的调控，如Pericentrin（Pcnt）样蛋白 (PLP)，一个保守的中心体支架也需要纤毛功能。完成本提案将 进一步了解plp mRNA的转录后调控。在人类中， 同源PCNT基因导致先天性疾病，如小头骨发育不良 原始侏儒II型（MOPD II）和21三体。类似地，在果蝇中，plp失调导致 多种缺陷，包括胚胎致死、神经元功能障碍和雄性不育。的机制 与PLP损失相关的多效性表型的根本原因还不完全清楚。预测人民党 编码12种mRNA变体，但是是什么机制导致了这些不同的RNA种类，以及它们如何在 表达的时空调控是完全未知的。在本K99/R 00建议书中，我将测试 假设PLP蛋白异构体的表达，再加上转录后调控plp mRNA在不同的组织中调节其不同的功能。提出了三个目标来检验这一点 假说.目的1：明确胚胎plp mRNA的定位和翻译机制。在目标2中， 我将探索不同启动子和3 'UTR使用对产生不同plp RNA的贡献 变体。在目标3中，我将确定PLP蛋白异构体的表达谱，并检查其生物学特性。 果蝇成神经细胞与早期胚胎中PLP亚型（包括PLPPM）的功能。完成 这项工作的结果将揭示空间和时间上不同的表达模式的功能机制， PLP蛋白在果蝇不同组织中的亚型，将提高我们对plp mRNA的理解 调节，其方面在人类疾病如神经变性病症中可被解除调节 和心血管疾病。此外，该奖项将提供RNA的技术和专业培训， 测序和生物信息学，CRISPR基因组编辑，以及先进的成像方法 我的专家指导。我将参加一个有组织的培训计划来提高我的专业能力 建立并运营我自己成功的独立实验室

PROJECT SUMMARY Centrosome deregulation is associated with developmental disorders, such as microcephaly, ciliopathy, and cardiovascular disease. Despite their fundamental importance to human health, relatively little is known about the regulation of genes encoding core centrosome components, such as Pericentrin (Pcnt)-like protein (PLP), a conserved centrosome scaffold also required for ciliary function. Completion of this proposal will advance our understanding of the post- transcriptional regulation of plp mRNA. In humans, deregulation of the homologous PCNT gene results in congenital diseases, such as microcephalic osteodysplastic primordial dwarfism type II (MOPD II) and Trisomy 21. Similarly, in Drosophila, plp deregulation leads to diverse defects, including embryonic lethality, neuron dysfunction, and male sterility. The mechanisms underlying the pleiotropic phenotypes associated with plp loss are incompletely understood. plp is predicted to encode 12 mRNA variants, but what mechanisms give rise to these distinct RNA species and how their expression is spatiotemporally regulated are completely unknown. In this K99/R00 proposal, I will test the hypothesis that the PLP protein isoform expression, coupled with the post- transcriptional regulation of plp mRNA, modulates its diverse functions within different tissues. Three aims are proposed to test this hypothesis. In Aim 1, I will identify mechanisms of embryonic plp mRNA localization and translation. In Aim 2, I will explore the contribution of alternative promoter and 3’UTR usage for the generation of different plp RNA variants. In Aim 3, I will determine the expression profile of PLP protein isoforms and examine the biological function of PLP isoforms, including PLPPM, in Drosophila neuroblasts versus early embryos. The completion of this work will reveal the mechanisms of spatially and temporally distinct expression patterns of functional PLP protein isoforms in different Drosophila tissues and will improve our understanding of plp mRNA regulation, aspects of which may be deregulated in human diseases, such as neurodegenerative disorders and cardiovascular disease. Moreover, this award will provide technical and professional training in RNA- sequencing and bioinformatics, CRISPR genome editing, and advanced imaging approaches under the guidance of my expert mentors. I will follow a structured training program to enhance my professional abilities to establish and run my own successful independent laboratory.