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

总结：现在很清楚，“epitranscriptome”，即，受调控核苷酸的模式和分布 mRNA的修饰是动态的，在大脑中具有功能性作用。我们在这一领域发挥了奠基作用， 开发了N6-甲基腺苷（m6 A）的转录组范围作图技术，这使我们能够 和其他人揭示了m6 A在不同组织，信号传导和疾病中的转录组动态 contexts.虽然m6 A被广泛研究，但它只是五种丰富的甲基修饰之一， 在20世纪70年代的mRNA中发现。其他四个是“扩展帽结构”的一部分，即 在mRNA的5'末端修饰的核苷酸。这些是m7 G帽上的甲基，2 '-O-甲基修饰 在mRNA中第一个和有时第二个转录核苷酸的核糖上，称为帽1和帽2， 分别最后，如果mRNA的第一个转录的核苷酸是腺苷，则它可以再甲基化一个。 核糖2 '-O-甲基化形成二甲基腺苷后的时间：N6，2'-O-二甲基腺苷（m6 Am）。其中， m6 Am和Cap 2的水平在组织之间变化，并显示出调节的证据。然而， 我们知道这些修饰的动态变化如何影响神经元中mRNA的命运。为了 揭示它们的功能，我们已经确定了合成m6 Am的酶，确定了第一个m6 Am阅读器， 并开发了一种在整个转录组中定位Cap 2的方法。为了显著提高 我们对神经元中cap epitranscriptome的动力学和功能的理解， 本研究的目的是：（1）揭示m6 Am在神经干细胞分化中的动力学机制。 m6 Am的动态调节的基础是未知的。为了了解哪些mRNA表现出动态和 调节水平的m6 Am，我们将使用我们的全转录组m6 Am作图技术来生成m6 Am的图谱。 m6 Am在不同的大脑区域。我们将确定指导m6 Am形成和调节的原则， 并确定这些动力学对神经干细胞分化是否重要。(2)以确定如何 m6 Am影响神经元mRNA的翻译和稳定性。为此，我们利用我们的 发现PCIF 1作为m6 Am形成甲基转移酶，以揭示m6 Am对翻译的影响， mRNA稳定性我们还将描述一个假定的m6 Am阅读器，以确定m6 Am如何 改变神经元mRNA。(3)以破译Cap 2表转录组的动力学和功能。我们 将获得Cap 2在整个大脑中的第一张地图。使用Cap 2地图和Cap 2的耗尽- 形成甲基转移酶，我们将确定帽2是否与改变mRNA的翻译，稳定性， RNA加工的其他方面。总的来说，这些研究将使我们能够绘制和确定 在神经元中控制mRNA命运和功能的“帽表转录组”。我们希望这项工作将 刺激了基因表达调控研究的新领域，重点是揭示信息如何编码 通过mRNA帽中的甲基修饰影响mRNA生物学。

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.