Mechanism and consequence of intron-retention in the adult brain

项目摘要 我们努力描绘将短暂体验转化为持久体验的生物化学过程， 记忆导致发现果蝇RNA结合蛋白Orb2A的功能性聚集对于 动物形成和保持记忆的能力。在上一个资助期，我们努力破译 通过研究Orb2A聚集的调控，我们发现Orb2A蛋白在成年人脑中的表达是 通过独特的机制控制。完全转录和多聚腺苷酸化的Orb2A转录物保留内含子 并且在果蝇脑中稳定表达。保留内含子的Orb2A mRNA不编码蛋白质，因为 到多个框内终止密码子然而，行为训练，产生长期的，但不是短期的， 记忆短暂地增加了编码剪接蛋白的mRNA水平。这种剪接事件，对长期的 记忆，是由一个特定的异构体的剪接调节Pasilla，psL。 内含子保留（Intron retention，IR）是一种研究较少的选择性剪接形式， 在一些实施方案中，细胞主要通过降解mRNA，将其捕获在细胞核中，从而减少蛋白质表达来抑制表达。 我们的研究结果表明，IR可以以一种新的方式使用：将mRNA保持在“平衡状态”，这可以允许 空间和时间整合各种信号，以急剧增加蛋白质表达来调节记忆。 这些观察使我们想知道，在成年果蝇的大脑中，IR有多普遍， IR在大脑中的分子基础以及经验如何与内含子保留机制相互作用以控制 蛋白质表达和记忆。我们已经从野生型果蝇脑中进行了poly（A）RNA测序。的 分析得出了一些令人惊讶的结果。首先，在所有可检测的内含子中，有8.9%对应于3031个基因， 显示在野生型果蝇脑中保留≥ 25%。其中包括55个与学习和记忆有关的基因。最近的一 一项全基因组研究显示，小鼠大脑中约6%的完全转录和多聚腺苷酸化的转录物也 保留了一个内含子值得注意的是，一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，聚集倾向的朊病毒样蛋白显著地 在内含子保留基因中过度表达。这包括一种新的朊病毒样磷酸酶，我们最近 鉴定从成人大脑中，我们已经确定了蛋白质结合保留内含子和相互作用的psL。在 目前的建议，我们打算调查如何记忆相关的刺激激活p38 a，一个假定的激酶psL， 行为刺激如何影响psL与其结合伴侣的相互作用，保留基序中的序列基序是什么 影响内含子保留，内含子保留如何控制聚集倾向蛋白表达，以及如何 这些过程中的每一个的扰动影响动物形成和保持记忆的能力。这些研究将 提供了深入了解IR介导的蛋白质表达控制，一种新的基因调控机制，以及 作为记忆和调节聚集倾向蛋白的分子基础。

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.