Control of cap-independent translation by N6-methyladenosine and FTO

“通过N6-甲基腺苷和FTO控制帽非依赖性翻译” 1 R01 CA186702-01 PI：Jaffrey，Samie R. 总结 癌细胞动态改变其蛋白质组成的能力是它们对不同类型的细胞应激（如DNA损伤和化疗）作出反应的主要机制。在许多情况下，细胞应激激活新的翻译模式，使特定蛋白质的表达，增强癌症表型。癌症特异性翻译控制各种蛋白质的表达，这些蛋白质在细胞存活和适应压力中发挥作用。mRNA的5 'UTR中的新序列元件和基序被认为在赋予转录物表现出癌症特异性翻译的能力方面具有关键作用。然而，它们的功能仍然神秘，因为它们通常缺乏序列保守性。此外，还不可能使用重建翻译起始的体外测定来概括它们的作用。在这个提议中，我们建议检查最近描述的mRNA碱基修饰，N[6]-甲基腺苷（m6 A）的功能，并确定它如何影响癌细胞中mRNA的翻译。此外，我们先前表明癌症风险基因，脂肪量和肥胖相关基因（FTO），选择性地使mRNA中的m6 A去甲基化，这表明它可能在调节细胞中的mRNA命运或翻译中发挥作用。为了显著地推进我们对这种调节蛋白质翻译的新途径的理解，我们的目标是：（1）开发SIMPL-Seq：第一种以单核苷酸分辨率定位m6 A的方法。虽然已知m6 A存在于约8，000种不同的mRNA中，但目前尚不可能确定其在mRNA中的精确位置。这对于突变这些残基和建立m6 A在翻译和其他过程中的功能是必要的。在这个目标中，我们使用一种新的化学生物学方法来映射，第一次，m6 A残基在整个转录组的不同的癌细胞系。(2)确定m6 A如何调节mRNA的翻译。为了理解m6 A影响翻译的机制，本研究的目的是：（1）含有m6 A的序列基序的序列和结构特征，使其能够影响癌细胞中观察到的每种类型的翻译起始;（2）影响核糖体募集到mRNA的m6 A结合蛋白;（3）m6 A在促进活细胞翻译中的作用。这些实验使用体外重建和基于细胞的实验来提供对m6 A介导的翻译调节的理解。(3)测试FTO作为翻译调节剂的作用。在这个目标中，我们将确定FTO是否是翻译的生理调节器。为此，我们将使用核糖体分析来鉴定通过不同的翻译起始模式翻译的转录本，并确定这些转录本是否受到FTO敲低或过表达的影响。这些实验将测试FTO是否具有“关闭”m6 A诱导的翻译的功能。总之，这三个目标的实验旨在解决长期存在的关于细胞中癌症特异性翻译途径调控的谜团。该提案描述了m6 A的功能作用，并测试了FTO是细胞中翻译途径的调节剂的想法。这些研究将重新定义我们对细胞中控制翻译的途径的基本理解，并为进一步探索m6 A和FTO在癌症和其他疾病中的作用提供基础。

“Control of cap-independent translation by N6-methyladenosine and FTO” 1 R01 CA186702-01 PI: Jaffrey, Samie R. SUMMARY The ability of cancer cells to dynamically change their protein composition is a major mechanism by which they respond to diverse types of cellular stress, such as DNA damage and chemotherapeutics. In many cases, cellular stresses activate novel modes of translation that enable the expression of specific proteins that enhance the cancer phenotype. Cancer-specific translation controls the expression of various proteins with roles in cell survival and adaptation to stress. Novel sequence elements and motifs in the 5’UTR of mRNAs are thought to have key roles in conferring the ability of transcripts to exhibit cancer-specific translation. However, their function remains mysterious because they typically lack sequence conservation. Moreover, it has not been possible to recapitulate their effects using in vitro assays that reconstitute translation initiation. In this proposal, we propose to examine the function of a recently described mRNA base modification, N[6]-methyladenosine (m6A), and determine how it affects translation of mRNAs in cancer cells. Furthermore, we previously showed that the cancer risk gene, fat mass and obesity associated gene (FTO), selectively demethylates m6A in mRNA, suggesting that it may have a role in regulating mRNA fate or translation in cells. In order to significantly advance our understanding of this novel pathway that regulates protein translation, our goals are: (1) To develop SIMPL-Seq: The first method to map m6A at single-nucleotide resolution. Although m6A is known to occur in ~8,000 different mRNAs, it is not currently possible to determine its precise location in mRNAs. This is necessary for mutating these residues and establishing the function of m6A in translation and other processes. In this aim we use a novel chemical biology approach to map, for the first time, m6A residues throughout the transcriptome of diverse cancer cell lines. (2) To determine how m6A regulates translation of mRNA. To understand the mechanism by which m6A influences translation, this aim will address: (1) the sequence and structural features of m6A-containing sequence motifs that enable it to affect each type of translation initiation seen in cancer cells; (2) the m6A-binding proteins that influence ribosome recruitment to mRNA; and (3) the role of m6A in promoting translation in living cells. These experiments use both in vitro reconstitution and cell-based experiments to provide an understanding of translational regulation mediated by m6A. (3) To test the role of FTO as a regulator of translation. In this aim, we will determine if FTO is a physiologic regulator of translation. To do this, we will use ribosome profiling to identify transcripts that are translated through diverse modes of translation initiation, and determine if these are affected by FTO knockdown or overexpression. These experiments will test whether FTO functions to “switch off” translation that is induced by m6A. Together, the experiments in these three aims seek to resolve the long-standing mystery regarding the regulation of cancer-specific translation pathways in cells. This proposal describes a functional role for m6A and tests the idea that FTO is a regulator of translation pathways in cells. These studies will redefine our basic understanding of pathways controlling translation in cells and provide a basis for further exploration of the role of m6A and FTO in cancer and other diseases.