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化过程 记忆导致发现果蝇RNA结合蛋白Orb2A的功能聚集对 动物形成和保持记忆的能力。在之前的拨款期间，在我们努力破译 对Orb2A聚集的调节，我们发现Orb2A蛋白在成人脑中的表达是 通过一种独特的机制进行控制。完全转录和多聚腺苷化的Orb2a转录本保留内含子 并在果蝇脑中稳定表达。保留内含子的Orb2AmRNA不能编码蛋白质，因为 到多个帧内停止密码子。然而，产生长期但不是短期的行为培训 记忆会瞬间增加编码拼接蛋白的信使核糖核酸水平。这一拼接事件，对长期而言非常重要 记忆，是由剪接调节蛋白PSL的特定亚型介导的。 内含子保留(IR)是一种研究较少的选择性剪接形式，被认为可以调节基因 主要是通过降解mRNA，将其困在细胞核中，从而减少蛋白质的表达。 我们的发现表明，IR可以以一种新的方式使用：将mRNA保持在一种稳定的状态，这可以允许 各种信号的空间和时间整合，急剧增加蛋白质表达，调节记忆。 这些观察使我们想知道IR在成年果蝇大脑中的普遍程度，以及 脑内IR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。我们已经从野生型苍蝇脑中进行了聚(A)RNA测序。这个 分析得出了一些令人惊讶的结果。首先，在所有可检测到的内含子中，有8.9%对应于3031个基因， 显示≥在野蝇脑中滞留了25%。其中包括55个与学习和记忆有关的基因。最近 全基因组研究显示，在小鼠大脑中，约6%的完全转录和多腺苷转录本也 保留一个内含子。值得注意的是，一些果蝇基因的小鼠同源基因，包括Orb2同源基因 CPEB2，也保留了内含子。第二，像Orb2一样，易于聚集的类病毒蛋白显著地 在内含子保留的基因中有过多的代表。这包括我们最近发现的一种新的普里恩样磷酸酶 已确认身份。从成人大脑中，我们已经识别出与保留的内含子结合并与PSL相互作用的蛋白质。在……里面 目前的建议是，我们打算研究记忆相关刺激如何激活P38A，P38A是PSL的一种可能的激酶， 行为刺激如何影响PSL与其结合伙伴的相互作用，保留的基序中有哪些序列基序 影响内含子保留、内含子保留如何控制聚集倾向蛋白的表达以及如何 这些过程中的每一个过程的干扰都会影响动物形成和保持记忆的能力。这些研究将 深入了解IR介导的蛋白质表达控制，这是一种新的基因调控机制 作为记忆和聚集倾向蛋白调节的分子基础。

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.