Isoform expression and post-transcriptional regulation of centrosomal plp mRNA

中心体 plp mRNA 的异构体表达和转录后调控

• 批准号: 10449716
• 负责人: Junnan Fang
• 金额: $ 9.97万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10449716
• 关键词: 3' Untranslated Regions; Award; Bioinformatics; Biological; Biological Assay; Biological Process; Biology; Brain; Cardiovascular Diseases; Cell physiology; Centrioles; Centrosome; Clustered Regularly Interspaced Short Palindromic Repeats; Congenital Disorders; Coupled; Data; Defect; Development; Disease; Down Syndrome; Down-Regulation; Drosophila genus; Embryo; Etiology; Functional disorder; Gene Expression; Gene Expression Regulation; Generations; Genes; Health; Human; Individual; Knowledge; Laboratories; Length; Male Sterility; Malignant Neoplasms; Mass Spectrum Analysis; Mentors; Messenger RNA; Microcephaly; Microtubule-Organizing Center; Microtubules; Modeling; Molecular Weight; Monitor; Mutate; Neurodegenerative Disorders; Neurodevelopmental Disorder; Neurons; Orthologous Gene; Pattern; Phase; Phenotype; Poly(A) Tail; Polyadenylation; Post-Transcriptional Regulation; Protein Isoforms; Proteins; RNA; RNA Splicing; RNA-Binding Proteins; Regulation; Reporter; Research Personnel; Role; Running; Structure; Technical Expertise; Testing; Testis; Tissues; Training; Training Programs; Transcript; Translations; Variant; Western Blotting; Work; cell motility; cell type; ciliopathy; developmental disease; differential expression; genome editing; human disease; imaging approach; improved; insight; neuroblast; neurogenesis; osteodysplastic primordial dwarfism; pericentrin; programs; promoter; protein function; recruit; scaffold; spatiotemporal; transcriptome sequencing; transcriptomics

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.

项目概要 中心体失调与发育障碍有关，例如小头畸形、纤毛病和 尽管心血管疾病对人类健康至关重要，但人们对其知之甚少。 关于编码核心中心体成分的基因的调控，例如 Pericentrin (Pcnt) 样蛋白 （PLP），纤毛功能也需要一个保守的中心体支架，完成该提案将。 我们对人类 plp mRNA 后期转录调控的理解， 同源PCNT基因导致先天性疾病，例如小头骨发育不良 II 型原始侏儒症 (MOPD II) 和 21 三体症。类似地，在果蝇中，plp 失调会导致 多种缺陷，包括胚胎致死、神经元功能障碍和雄性不育。 与 plp 损失相关的多效性表型背后的预测尚不完全清楚。 编码 12 种 mRNA 变体，但是是什么机制产生了这些不同的 RNA 种类以及它们是如何产生的 表达是否受时空调控是完全未知的。在这个 K99/R00 提案中，我将测试 假设 PLP 蛋白亚型表达，与 plp 的转录后调控相结合 mRNA 在不同组织内调节其不同功能，提出了三个目标来测试这一点。 在目标 1 中，我将确定胚胎 plp mRNA 定位和翻译的机制。 我将探讨替代启动子和 3’UTR 使用对生成不同 plp RNA 的贡献 在目标 3 中，我将确定 PLP 蛋白亚型的表达谱并检查生物学特性 PLP亚型（包括PLPPM）在果蝇神经母细胞与早期胚胎中的功能完成。 这项工作将揭示功能性蛋白质在空间和时间上不同的表达模式的机制 不同果蝇组织中的 PLP 蛋白亚型将提高我们对 plp mRNA 的理解 调节，在人类疾病中可能会放松调节，例如神经退行性疾病 此外，该奖项还将提供 RNA- 领域的技术和专业培训。 测序和生物信息学、CRISPR 基因组编辑以及先进的成像方法 我将遵循结构化的培训计划来提高我的专业能力。 建立并经营我自己成功的独立实验室。