The cap epitranscriptome: Regulation of mRNA fate and function by cap-associated methyl modifications

帽子表观转录组：帽子相关甲基修饰对 mRNA 命运和功能的调节

• 批准号: 10606589
• 负责人: SAMIE R JAFFREY
• 金额: $ 54.7万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10606589
• 关键词: Adenosine; Affect; Area; Biology; Brain; Brain region; Cell Proliferation; Cells; Data Set; Disease; Enzymes; Exhibits; FRAP1 gene; Gene Expression Regulation; Genetic Transcription; Maps; Mediating; Messenger RNA; Methods; Methylation; Methyltransferase; Modification; Neurons; Nucleotides; Pattern; Poly(A)+ RNA; Protein Isoforms; RNA Processing; Reader; Regulation; Research; Ribose; Role; Signal Transduction; Site; Structure; Switch Genes; Techniques; Technology; Testing; Time; Tissues; Transcript; Transcription Initiation; Transcription Initiation Site; Translations; Work; cell growth; cohort; epitranscriptome; epitranscriptomics; experimental study; insight; mRNA Stability; mRNA Translation; mRNA capping; nerve stem cell; novel; stem cell differentiation; transcriptome

SUMMARY: It is now clear that the “epitranscriptome,” i.e., the pattern and distribution of regulated nucleotide modifications in mRNA, is dynamic and has functional roles in the brain. We had a founding role in this field by developing the technology for transcriptome-wide mapping of N6-methyladenosine (m6A), which allowed us and others to reveal the transcriptome-wide dynamics of m6A in diverse tissues, signaling and disease contexts. Although m6A is widely studied, it is only one of five abundant methyl modifications that were discovered in mRNA in the 1970's. The other four are part of the “extended cap structure,” i.e. the cluster of modified nucleotides at the 5' end of mRNA. These are the methyl on the m7G cap, 2'-O-methyl modifications on the ribose of the first and sometimes the second transcribed nucleotides in mRNA, called Cap 1 and Cap 2, respectively. Lastly, if the first transcribed nucleotide of an mRNA is adenosine, it can be methylated one more time after ribose 2'-O-methylation to form dimethyladenosine: N6,2'-O-dimethyladenosine (m6Am). Of these, levels of m6Am and Cap 2 vary between tissues and show evidence for regulation. Nevertheless, little is known about how these dynamic changes in these modifications affects mRNA fates in neurons. In order to uncover their function, we have identified the enzyme that synthesizes m6Am, identified the first m6Am reader and developed a method for mapping Cap 2 throughout the transcriptome. In order to significantly advance our understanding of the dynamics and function of the cap epitranscriptome in neurons, the specific aims of this proposal are: (1) To uncover the mechanism for m6Am dynamics in neural stem cell differentiation. The basis for the dynamic regulation of m6Am is unknown. To understand which mRNAs exhibit dynamic and regulated levels of m6Am, we will use our transcriptome-wide m6Am mapping technique to generate maps of m6Am in different brain regions. We will determine the principles that guide m6Am formation and regulation, and determine if these dynamics are important for neural stem cell differentiation. (2) To determine how m6Am affects the translation and stability of neuronal mRNA. In this aim, we take advantage of our discovery of PCIF1 as the m6Am-forming methyltransferase to uncover the effects of m6Am on translation and mRNA stability. We will also characterize a putative m6Am reader, to identify a mechanism for how m6Am alters neuronal mRNAs. (3) To decipher the dynamics and function of the Cap 2 epitranscriptome. We will obtain the first maps of Cap 2 throughout the brain. Using the Cap 2 maps and depletion of the Cap2- forming methyltransferase, we will determine if Cap 2 is associated with altered mRNA translation, stability, or other aspects of RNA processing. Overall, these studies will allow us to map and determine the role of the “cap epitranscriptome” in controlling mRNA fate and function in neurons. We expect that this work will stimulate a new area of gene expression regulation research focusing on uncovering how information encoded by methyl modifications in mRNA caps influences mRNA biology.

摘要：现在很明显，“表面翻译”，即受监管的核苷酸的模式和分布 mRNA的修饰是动态的，并且在大脑中具有功能作用。我们通过 开发N6-甲基二苯胺（M6A）的整个转录组图的技术，这使我们 以及其他以揭示多样性组织，信号和疾病中M6a的全转录组动力学的 contexts。尽管M6A已广泛研究，但它只是五种丰富的甲基修饰之一 在1970年代在mRNA中发现。其他四个是“扩展盖结构”的一部分，即 mRNA的5'末端的修饰核动肽。这些是M7G帽上的甲基，2'-O-甲基修饰 在第一个和有时是第二次转录的mRNA中的核苷酸的核糖上，称为cap 1和cap 2， 最后，如果mRNA的第一个转录核苷酸是腺苷，则可以甲基化一个 核糖2'-O-甲基化后的时间形成二甲基二胺：N6,2'-O-二甲基二胺（M6AM）。其中， M6AM和2的水平在组织之间有所不同，并显示了调节的证据。然而，几乎没有 这些修饰中的这些动态变化如何影响神经元中的mRNA命运。为了 发现它们的功能，我们已经确定了合成M6AM的酶，确定了第一个M6AM读取器 并开发了一种在整个转录组中绘制CAP 2的方法。为了显着前进 我们对神经元中CAP上表突出组的动态和功能的理解，是我们的特定目的 该建议是：（1）发现神经干细胞分化中M6AM动力学的机制。 M6AM动态调控的基础尚不清楚。了解哪些mRNA暴露了动态和 受监管的M6am级别，我们将使用全转录组的M6AM映射技术来生成地图 M6am在不同的大脑区域。我们将确定指导M6AM形成和法规的原则， 并确定这些动力学对于神经干细胞分化是否重要。 （2）确定如何 M6AM影响神经元mRNA的翻译和稳定性。在这个目标中，我们利用了我们的优势 发现PCIF1作为M6AM形成的甲基转移酶，以发现M6AM对翻译和翻译的影响 mRNA稳定性。我们还将表征推定的M6AM阅读器，以确定M6AM的机制 改变神经元mRNA。 （3）解读CAP 2表面参考组的动力学和功能。我们 将在整个大脑中获得CAP 2的第一张地图。使用CAP 2地图和CAP2-的部署 形成甲基转移酶，我们将确定CAP 2是否与mRNA翻译，稳定性或 RNA处理的其他方面。总体而言，这些研究将使我们能够映射并确定 控制神经元中mRNA命运和功能的“ CAP同源转录组”。我们希望这项工作将 刺激基因表达调节研究的新领域，重点是发现信息如何编码 通过mRNA帽中的甲基修饰会影响mRNA生物学。