Illuminating the gene regulation underlying meiotic differentiation

阐明减数分裂分化背后的基因调控

• 批准号: 10320392
• 负责人: Gloria Ann Brar
• 金额: $ 43.37万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-01-01 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10320392
• 关键词: Address; Case Study; Cell physiology; Cells; Cellular Structures; Codon Nucleotides; Event; Gene Expression; Gene Expression Regulation; Genes; Genome; Genomic approach; Germ Cells; Label; Meiosis; Messenger RNA; Metabolic; Modeling; Molecular; Open Reading Frames; Organism; Process; Production; Protein Isoforms; Proteins; Regulation; Regulator Genes; Reporter; Research; Role; Saccharomycetales; Sexual Reproduction; Structure; Time; Transcript; Translating; Translation Initiation; Translations; Work; base; expectation; experimental study; mRNA Transcript Degradation; precursor cell; programs

ABSTRACT Meiosis is the conserved differentiation program that is responsible for gamete formation. As a cell progress through meiotic differentiation, it undergoes unidirectional changes in cellular structure and function that are largely driven by gene expression changes. Because the molecular basis for most meiotic transitions remains mysterious, my lab aims to illuminate the gene regulatory circuitry that programs meiotic differentiation. We use budding yeast to study this process because well studied meiotic factors are highly conserved and because this organism uniquely offers access to the large number of highly synchronous cells that is key to genomic approaches that we routinely employ. Our studies have enabled identification of proteins involved in key meiotic processes, and new regulatory events during meiosis. These studies have also uncovered major surprises in the genes that meiotic cells express and how they regulate these genes. Among these surprises, we found an unconventional mode of gene regulation, involving regulated toggling between a translatable mRNA isoform and one that is 5’ extended and poorly translated, to be commonly used to drive meiotic protein levels over time. We have found this mode of regulation to be important in meiosis but also in other conditions, and a major focus of our research is to better understand how it works. Although we know that upstream open reading frames (uORFs) are responsible for repressed ORF translation on some extended mRNA isoforms, we do not know why this is not true of all cases. We will address this question using reporter experiments, and analysis of mRNA structures and sequences of repressed versus non-repressed transcripts. We also do not understand how mRNA degradation impacts this regulation and meiotic gene expression more broadly, which we will study using new metabolic labeling approaches. Beyond unconventional regulation of known genes, we also discovered that meiotic cells translate many genes were not previously identified. These include hundreds of genes that are translated starting with non-AUG codons, and thousands that are shorter than the 100 codon cutoff that was used to annotate genomes. We have validated the expression of these non-canonical proteins and are now studying the molecular mechanisms underlying their synthesis and their specific cellular roles. We are investigating why non-AUG translation initiation is common in meiosis, primarily using study of candidate regulatory factors that we have identified. We are performing pooled screens to identify roles for the many short meiotic proteins, and directed study of cases in which the short proteins include domains of characterized proteins. Together the projects proposed here will explain how and why meiotic cells employ non-canonical gene regulatory features, which we believe is critical to unraveling the molecular control of meiotic progression.

抽象的 减数分裂是负责配子形成的保守分化程序。作为一个细胞 通过减数分裂分化，细胞结构和功能发生单向变化 这很大程度上是由基因表达变化驱动的。因为大多数减数分裂转变的分子基础 仍然神秘，我的实验室旨在阐明编程减数分裂的基因调控电路 差异化。我们使用芽殖酵母来研究这个过程，因为经过充分研究的减数分裂因子高度 保守，因为这种生物体独特地提供了大量高度同步的细胞 这是我们常规采用的基因组方法的关键。我们的研究已经能够鉴定蛋白质 参与关键的减数分裂过程以及减数分裂期间的新调控事件。这些研究还 揭示了减数分裂细胞表达的基因以及它们如何调节这些基因的重大惊喜。 在这些惊喜中，我们发现了一种非常规的基因调控模式，涉及调控切换 介于可翻译的 mRNA 异构体和 5' 延伸且翻译不良的 mRNA 异构体之间，常用 随着时间的推移提高减数分裂蛋白水平。我们发现这种调节模式在减数分裂中很重要，但是 在其他条件下也是如此，我们研究的一个主要重点是更好地了解它是如何工作的。虽然我们 知道上游开放阅读框 (uORF) 负责抑制某些 ORF 翻译 扩展的 mRNA 异构体，我们不知道为什么并非所有情况都是如此。我们将使用以下方法来解决这个问题 记者实验，以及抑制与非抑制的 mRNA 结构和序列分析 成绩单。我们也不了解 mRNA 降解如何影响这种调节和减数分裂基因 更广泛的表达，我们将使用新的代谢标记方法进行研究。 除了已知基因的非常规调控之外，我们还发现减数分裂细胞翻译 许多基因以前未被识别。其中包括数百个基因，这些基因从以下位置开始翻译： 非 AUG 密码子，以及数千个比用于注释的 100 个密码子截止值短的密码子 基因组。我们已经验证了这些非典型蛋白质的表达，现在正在研究 它们的合成及其特定细胞作用的分子机制。我们正在调查原因 非 AUG 翻译起始在减数分裂中很常见，主要利用对候选调控因子的研究 我们已经确定了。我们正在进行汇总筛选，以确定许多短减数分裂蛋白的作用，并且 对短蛋白包含特征蛋白结构域的情况进行定向研究。一起 这里提出的项目将解释减数分裂细胞如何以及为何采用非规范基因调控 我们认为这对于揭示减数分裂进程的分子控制至关重要。