Mechanism and consequence of intron-retention in the adult brain

成人大脑内含子保留的机制和后果

• 批准号: 10569524
• 负责人: Kausik Si
• 金额: $ 41.25万
• 依托单位: STOWERS INSTITUTE FOR MEDICAL RESEARCH
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10569524
• 关键词: Acute; Address; Adult; Alternative Splicing; Animals; Behavioral; Behavioral Assay; Binding; Binding Proteins; Biochemical Process; Biological Assay; Brain; Cell Nucleus; Code; Cues; Disease; Drosophila genus; Event; Excision; Food; Gene Expression; Genes; Genetic Transcription; Grant; Hunger; Immunoprecipitation; Impairment; Introns; Large-Scale Sequencing; Lead; Learning; Maintenance; Measures; Mediating; Memory; Messenger RNA; Molecular; Mus; Mutate; Mutation; Nervous System; Nervous System Physiology; Neurons; Nucleotides; Odors; Organism; Pathway interactions; Phosphoproteins; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Physiological; Poly A; Poly(A)+ RNA; Polyadenylation; Process; Protein Isoforms; Protein Splicing; Proteins; Proteomics; Quality Control; RNA; RNA Splicing; RNA-Binding Proteins; Regulation; Regulator Genes; Reporting; Role; Sensory; Signal Transduction; Site; Stimulus; Synaptosomes; Terminator Codon; Training; Trans-Activators; Transact; Transcript; candidate identification; cis acting element; crosslink; experience; fly; genetic manipulation; genome-wide analysis; insight; long term memory; memory retention; mutant; novel; p38 Mitogen Activated Protein Kinase; prevent; prion-like; protein aggregation; protein expression; sensory stimulus; synaptic function; tool; transcriptome; transcriptome sequencing

Project Summary Our efforts to delineate the biochemical processes that convert a transient experience to a persistent memory led to the discovery that functional aggregation of a Drosophila RNA-binding protein Orb2A is critical for the animals’ ability to form and retain a memory. During the previous grant period, in our effort to decipher regulation of Orb2A aggregation, we discovered that the expression of Orb2A protein in the adult brain is controlled via a unique mechanism. The fully transcribed and polyadenylated Orb2A transcripts retain an intron and are stably expressed in the fly brain. The intron-retained Orb2A mRNA does not code for a protein, owing to multiple in frame stop codons. However, behavioral training that produces long-term but not short-term memory transiently increases the spliced protein-coding mRNA level. This splicing event, important for long-term memory, is mediated by a specific isoform of the splicing regulator Pasilla, psL. Intron retention (IR), a poorly studied form of alternative splicing, was thought to modulate gene expression primarily by degrading the mRNA, trapping it in the nucleus and thereby reducing protein expression. Our findings suggest that IR can be used in a novel way: to hold mRNA in a ‘poised state’, which can allow spatial and temporal integration of various signals to acutely increase protein expression to regulate memory. These observations led us to ask how prevalent IR is in the adult Drosophila brain, and what is the molecular basis of IR in the brain and how experience interacts with the intron-retention mechanisms to control protein expression and memory. We have performed poly(A) RNA-sequencing from wild type fly brains. The analysis provided some surprising results. First, 8.9% of all detectable introns, corresponding to 3031 genes, show ≥ 25% retention in wildtype fly brain. These includes 55 genes with role in learning and memory. A recent genome-wide study revealed ~6% of fully transcribed and polyadenylated transcripts in the mouse brain also retains an intron. Remarkably, the mouse orthologues of some the Drosophila genes, including Orb2 orthologue CPEB2, also retains intron. Second, like Orb2, aggregate-prone prion-like proteins are significantly overrepresented in the intron retained genes. This includes a novel prion-like phosphatase we have recently identified. From adult brain, we have identified proteins that bind to a retained intron and interacts with psL. In the current proposal we intend to investigate how memory-related stimuli activate p38a, a putative kinase of psL, how behavioral stimuli effect psL interaction with its binding partners, what sequence motifs in a retained motif influence intron retention, how intron retention controls expression of aggregate-prone proteins and how perturbation of each of these processes effects animals ability to form and retain memory. These studies would provide insight into the IR-mediated control of protein expression, a novel gene regulatory mechanism, as well as the molecular basis of memory and the regulation of aggregate-prone proteins.

项目摘要 我们为描述将瞬态体验转化为持续的生化过程的努力 记忆导致发现果蝇RNA结合蛋白ORB2A的功能汇总对于 动物的形成和保留记忆的能力。在上一个赠款期间，我们努力破译 ORB2A聚集的调节，我们发现ORB2A蛋白在成人大脑中的表达是 通过独特的机制控制。完全转录和聚腺苷酸化的ORB2A转录物保留了内含子 并在苍蝇大脑中稳步表达。内含子保留的ORB2A mRNA不为蛋白质编码， 到多个框架停止密码子。但是，行为训练会产生长期但不产生短期的行为训练 记忆会瞬时增加剪接的蛋白质编码mRNA水平。这个拼接事件，对于长期很重要 记忆是由剪接调节器Pasilla（PSL）的特定同工型介导的。 内含子保留（IR）是一种替代剪接形式不良的形式，被认为是调节基因的 表达主要通过降解mRNA，将其捕获到核中，从而降低蛋白质的表达。 我们的发现表明，IR可以以新颖的方式使用：将mRNA保持在“昂贵的状态”中，这可以允许 各种信号的空间和临时整合以急性增加蛋白质表达以调节记忆。 这些观察结果使我们询问了成年果蝇大脑中的IR流行程度，什么是 IR在大脑中的分子基础以及经验如何与控制内含子的机制相互作用 蛋白质表达和记忆。我们已经从野生型飞脑进行了poly（a）RNA测序。这 分析提供了一些令人惊讶的结果。首先，所有可检测的内含子中的8.9％，对应于3031个基​​因 在野生型蝇脑中显示≥25％的保留率。其中包括55个在学习和记忆中作用的基因。最近 全基因组的研究表明，在小鼠脑中的完全转录和多腺苷酸化转录本也有约6％ 值得注意的是，某些果蝇基因的小鼠直系同源物，包括ORB2直系同源物 CPEB2，也保留内含子。第二，像ORB2一样，易于骨料的prion样蛋白质显着 内含子保留基因的代表过多。这包括我们最近有一个新型的prion样磷酸酶 确定。从成人大脑中，我们发现了与保留内含子结合并与PSL相互作用的蛋白质。 当前的建议我们打算研究与记忆有关的刺激如何激活p38a，p38a是PSL的推定激酶， 行为刺激如何影响PSL与其结合伴侣的相互作用，保留基序中哪些序列基序 影响内含子保留，内含子保留如何控制易蛋白质的表达以及如何 这些过程中每个过程的扰动都会影响动物形成和保留记忆的能力。这些研究会 还深入了解IR介导的对蛋白质表达的控制，这是一种新型的基因调节机制 作为记忆的分子基础和调节骨料蛋白的调节。