RNA Control of Neural Function

我们的工作集中在神经功能的转录后控制，重点是神经元的翻译控制。 突触可塑性和学习记忆。我们研究了神经发育和神经变性 翻译出错时出现的混乱。我们发现，错误调节的翻译会导致 选择性剪接和RNA降解，这反过来又有助于神经病理学。更具体地说，我们的 研究由三个不同但互补的领域组成：（1）CPEB 1和细胞质多聚腺苷酸化 （2）FMRP对翻译的调控，重点是核糖体停滞和密码子使用; (3)选择性剪接的FMRP调节。CPEB 1调节的胞质多聚腺苷酸化控制翻译 在突触后区室，这反过来又改变突触强度，学习的基础细胞基础 和记忆我们实验室的分子、电生理和行为实验 证明CPEB 1调节活性依赖性胞质多聚腺苷酸化诱导的翻译， 进而改变突触强度和认知。CPEB 1使几种促进poly（A）尾的蛋白质成核 生长和移除，并介导翻译起始;它们还调节可塑性和动物行为。另一 对大脑功能重要的RNA结合蛋白是FMRP，脆性X综合征基因FMR 1的产物。 FMRP在大脑中结合>1000个RNA并调节翻译，主要通过阻止核糖体易位到 特定的mRNA。一种这样的mRNA编码表观遗传因子SETD 2，其催化染色质标记 H3K36me3.在FMRP缺陷小鼠脑中，SETD 2水平升高，H3 K36 me 3染色质 主要位于基因体中的景观被破坏。H3 K36 me 3与替代前mRNA连接 在FMRP敲除（KO）小鼠中存在广泛的剪接错误调节。主要目标 我们未来的研究将解决关于RNA调节神经功能的关键未解问题， 主要使用小鼠模型：（a）CPEB 1缺陷挽救FMRP KO小鼠中的脆性X病理生理学。 这种拯救是否涉及核糖体停滞和/或多聚腺苷酸化？(b)CPEB 1还调节3 'UTR长度。 这种情况发生的机制是什么？(c)FMRP如何阻止特定mRNA上的核糖体？并 FMRP作为核糖体转运的分子路障和/或FMRP与核糖体相互作用？(d)如何 FMRP调节可变剪接吗？一些错误剪接事件似乎涉及H3 K36 me 3，但 这是少数。FMRP是否调节编码剪接因子的mRNA的翻译，和/或 FMRP是一种穿梭蛋白，它是否直接影响剪接？(e)FMRP是如何利用密码子最优性 调节翻译和RNA稳定性？我们将通过核糖体分析来解决这些问题，我们修改了核糖体分析， 区分易位和停滞的核糖体，以及直接的纳米孔RNA测序， 在近核苷酸分辨率下的poly（A）尾大小以及通过选择性剪接产生的RNA同种型。我们将 从小鼠大脑中耗尽关键调节因子，并评估突触功能和动物行为。

Our work focuses on post-transcriptional control of neural function with emphasis on translational control of synaptic plasticity and learning and memory. We investigate the neurodevelopmental and neurodegenerative disorders that arise when this translation goes awry. We find that mis-regulated translation leads to changes in alternative splicing and RNA degradation, which in turn contribute to neuropathology. More specifically, our research is comprised of three distinct but complementary areas: (1) CPEB1 and cytoplasmic polyadenylation control of translation; (2) FMRP regulation of translation with emphasis on ribosome stalling and codon usage; (3) FMRP regulation of alternative splicing. CPEB1-regulated cytoplasmic polyadenylation governs translation in post-synaptic compartments, which in turn modifies synaptic strength, the underlying cellular basis of learning and memory. Molecular, electrophysiological, and behavioral experiments from our laboratory have demonstrated that CPEB1 regulates activity-dependent cytoplasmic polyadenylation-induced translation, which in turn modifies synaptic strength, and cognition. CPEB1 nucleates several proteins that promote poly(A) tail growth and removal and mediate translation initiation; they also regulate plasticity and animal behavior. Another RNA binding protein important for brain function is FMRP, the product of the Fragile X Syndrome gene FMR1. FMRP binds >1000 RNAs in the brain and regulates translation, primarily by stalling ribosome translocation on specific mRNAs. One such mRNA encodes the epigenetic factor SETD2, which catalyzes the chromatin mark H3K36me3. In FMRP-deficient mouse brain, SETD2 levels are elevated and the H3K36me3 chromatin landscape, which is principally located in gene bodies, is disrupted. H3K36me3 is linked to alternative pre-mRNA splicing and there is widespread mis-regulation of splicing in FMRP knockout (KO) mice. The main objectives our research going forward will address key unanswered questions regarding RNA regulation of neural function, primarily using mouse models: (a) CPEB1-deficiency rescues Fragile X pathophysiology in FMRP KO mice. Does this rescue involve ribosome stalling and/or polyadenylation? (b) CPEB1 also regulates 3’UTR length. What is the mechanism by which this occurs? (c) How does FMRP stall ribosomes on specific mRNAs? Does FMRP act as a molecular roadblock to ribosome transit and/or does FMRP interact with the ribosome? (d) How does FMRP regulate alternative splicing? Some of the mis-splicing events appear to involve H3K36me3, but these are in the minority. Does FMRP regulate the translation of mRNAs encoding splicing factors, and/or does FMRP, which is a shuttling protein, affect splicing directly? (e) How does FMRP employ codon optimality to regulate translation and RNA stability? We will address these issues by ribosome profiling, which we modified to distinguish between translocating and stalled ribosomes, and direct nanopore RNA sequencing, which yields poly(A) tail size at near-nucleotide resolution as well as RNA isoforms that arise by alternative splicing. We will deplete key regulatory factors from the mouse brain and assess synaptic function and animal behavior.