RNA Control of Neural Function

RNA 控制神经功能

• 批准号: 10622122
• 负责人: Joel D Richter
• 金额: $ 42.72万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-01 至 2028-02-29
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10622122
• 关键词: 3' Untranslated Regions; Ablation; Affect; Alternative Splicing; Animal Behavior; Area; Behavioral; Binding; Brain; Chromatin; Codon Nucleotides; Cognition; Cytoplasm; Electrophysiology (science); Epigenetic Process; Event; Excision; FMR1; FMRP; Fragile X Syndrome; Functional disorder; Genes; Genetic Transcription; Growth; Health; Human; Knockout Mice; Laboratories; Lead; Learning; Length; Link; Mediating; Memory; Messenger RNA; Minority; Molecular; Mus; Neurodegenerative Disorders; Neurodevelopmental Disorder; Neurophysiology - biologic function; Nucleotides; Poly A; Poly(A) Tail; Polyadenylation; Post-Transcriptional Regulation; Protein Isoforms; Proteins; RNA; RNA Degradation; RNA Splicing; RNA Stability; RNA-Binding Proteins; Regulation; Research; Resolution; Ribosomes; Synapses; Synaptic plasticity; Translation Initiation; Translational Regulation; Translations; Work; autism spectrum disorder; experimental study; mRNA Precursor; mRNA Translation; mouse model; nanopore; neuropathology; neuroregulation; postsynaptic; ribosome profiling; synaptic function; transcriptome sequencing

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.

我们的工作着重于对神经功能的转录后控制，重点是翻译控制 突触可塑性，学习和记忆。我们研究神经发育和神经退行性 当这种翻译出现问题时会出现的疾病。我们发现未经调节的翻译会导致变化 替代剪接和RNA降解，进而有助于神经病理学。更具体地说，我们的 研究完成了三个不同但互补的领域：（1）CPEB1和细胞质聚腺苷酸化 控制翻译； （2）FMRP对翻译的调节，重点是核糖体停滞和密码子使用； （3）FMRP调节替代剪接。 CPEB1调节的细胞质聚腺苷酸化控制翻译 在突触后隔室中，这又改变了突触强度，这是学习的基础基础 和内存。我们实验室的分子，电生理和行为实验 证明CPEB1调节活性依赖性细胞质聚腺苷酸化诱导的翻译，这是 反过来又改变了突触强度和认知。 CPEB1核能促进poly（a）尾巴的几种蛋白质 生长和去除和中介翻译计划；它们还调节可塑性和动物行为。其他 RNA结合蛋白对脑功能很重要的是FMRP，这是脆弱的X综合征基因FMR1的乘积。 FMRP在大脑中绑定> 1000 RNA并调节翻译，主要是在核糖体翻译上停滞 特定的mRNA。一种这样的mRNA编码表观遗传因子setD2，它催化染色质标记 H3K36me3。在缺乏FMRP的小鼠脑中，SETD2水平升高，H3K36ME3染色质 景观主要位于基因体中，是残疾人的。 H3K36me3与替代前MRNA有关 剪接，FMRP敲除（KO）小鼠中的剪接存在广泛的不调节。主要目标 我们未来的研究将解决有关神经功能的RNA调节的关键问题， 使用小鼠模型的主要：（a）FMRP KO小鼠中的CPEB1缺乏响应脆弱的X病理生理学。 这种救援是否涉及核糖体停滞和/或聚腺苷酸化？ （b）CPEB1还调节3'UTR长度。 发生这种情况的机制是什么？ （c）FMRP失速核糖体在特定mRNA上如何？做 FMRP充当核糖体传输的分子障碍和/或FMRP是否与核糖体相互作用？ （d）如何 FMRP是否调节替代剪接？一些失误的事件似乎涉及H3K36me3，但是 这些是少数派。 FMRP是否调节编码剪接因子的mRNA翻译和/或确实 FMRP是一种穿梭蛋白的FMRP直接影响剪接？ （e）FMRP员工密码子如何最佳 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，我们将其修改为 区分易位和失速核糖体和直接纳米孔RNA测序，该测序产生 近核苷酸分辨率以及通过替代剪接产生的RNA同工型的poly（a）尾巴尺寸。我们将 从小鼠大脑和评估突触功能和动物行为中耗尽关键调节因素。