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

摘要：现在已经很清楚了，“表位转录组”，即受调控的核苷酸的模式和分布 信使核糖核酸的修饰是动态的，在大脑中具有功能作用。我们在这一领域起到了开创性的作用 开发N6-甲基腺苷(M6A)的转录组全图谱技术，这使得我们能够 和其他人揭示了m6A在不同组织、信号和疾病中的转录组范围的动态 上下文。尽管M6A被广泛研究，但它只是五种丰富的甲基修饰之一 另外四个是“延伸帽结构”的一部分，即 M RNA 5‘端的修饰核苷酸。这些是m7G帽上的甲基，2‘-O-甲基修饰 在信使核糖核酸的第一个，有时是第二个转录的核苷酸的核糖上，称为第一和第二帽， 分别进行了分析。最后，如果一个信使核糖核酸的第一个转录的核苷酸是腺苷，它可以再甲基化一个。 核糖2‘-O-甲基化形成二甲基腺苷的时间：N6，2’-O-二甲基腺苷(M6Am)。其中， M6Am和Cap 2的水平因组织而异，并显示出调控的证据。然而，几乎没有什么是 了解这些修饰的动态变化如何影响神经元中的mRNA命运。为了 揭开了它们的功能，我们鉴定了合成m6Am的酶，鉴定了第一个m6Am阅读器 并开发了一种在整个转录组中定位第二帽的方法。为了显著地推进 我们对神经元中CAP表位编码组的动力学和功能的理解，以及 这一建议是：(1)揭示m6Am在神经干细胞分化中的动力学机制。 M6Am的动态调节基础尚不清楚。为了了解哪些mRNA表现出动态和 ，我们将使用我们的转录组范围的m6Am图谱技术来生成m6Am的图谱 M6AM存在于不同的脑区。我们将确定指导M6AM形成和监管的原则， 并确定这些动力学对神经干细胞分化是否重要。(2)决定如何 M6AM影响神经元mRNA的翻译和稳定性。在这个目标中，我们利用我们的 发现PCIF1作为m6Am形成甲基转移酶以揭示m6Am对翻译和 信使核糖核酸稳定性。我们还将描述一个假定的m6Am阅读器，以确定m6Am如何 改变神经元的mRNAs。(3)破译第2章表位编码组的动力学和功能。我们 将在整个大脑中获得第一张第二帽的地图。使用第2章的地图和第2章的耗尽- 形成甲基转移酶，我们将确定第二帽是否与改变的mRNA翻译、稳定性或 RNA加工的其他方面。总体而言，这些研究将使我们能够绘制和确定 “帽表位转录组”在神经元中控制信使核糖核酸的命运和功能。我们预计这项工作将 推动基因表达调控研究的一个新领域，重点是揭示信息是如何编码的 通过对信使核糖核酸帽的甲基化修饰来影响信使核糖核酸的生物学。

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.