Live-cell single-molecule investigation of localization and fate of m6A-modified mRNA

转录后RNA修饰对细胞活力至关重要。今天，已知蛋白质编码以及非编码RNA中的超过170种转录后RNA修饰。一种这样的RNA修饰是mRNA（N6-甲基腺苷，m6 A）和非编码RNA中腺嘌呤嘌呤环第六位的甲基化。m6 A代表真核生物中最丰富的内部mRNA修饰。m6 A修饰对mRNA代谢的许多方面至关重要，包括核前mRNA加工、mRNA的胞质输出、mRNA翻译、mRNA衰变和与RNA颗粒的相互作用。RNA测序研究发现，m6 A修饰在mRNA的终止密码子附近和3'非翻译区（3' UTR）中高度富集。3' UTR影响mRNA的稳定性、翻译效率及其亚细胞定位。生物学上重要的和功能性的甲基化靶3' UTR的一个实例由性别决定区y-盒2（SOX 2）mRNA给出，其编码关键转录因子并代表致癌基因。其甲基化状态分别显著地塑造mRNA的命运和细胞的活力。然而，关于3' UTR m6 A修饰的mRNA及其对翻译效率、动力学和核糖体负载以及RNA颗粒相互作用的影响的一些基本问题仍然没有答案。利用SOX 2 mRNA的3' UTR作为模型m6 A系统，结合尖端的基于显微注射的单分子RNA跟踪方法，mRNA翻译可视化技术和基于超高分辨率活细胞的单分子荧光显微镜，该项目的目的是表征mRNA中3' UTR甲基化对翻译效率，翻译动力学，以及在正常和应激条件下其向RNA颗粒（P体/应激颗粒）的分配动力学以及在RNA颗粒中的富集。所有这些将为理解mRNA中m6 A修饰提供前所未有的见解，并为基因调控过程的动力学（统称为epitranscriptomics）打开一个新的视角和概念。

Post-transcriptional RNA modifications are crucial for cell viability. Today over 170 posttranscriptional RNA modifications in protein coding as well as non-coding RNA are known. One such RNA modification is methylation on the sixth position of the purine ring of adenine in both mRNA (N6-methyladenosine, m6A) and non-coding RNA. m6A represents the most abundant internal mRNA modification in eukaryotes. Its importance is highlighted by the fact that m6A modification is crucial for many aspects of mRNA metabolism from birth to death that includes nuclear pre mRNA processing, cytosolic export of mRNA, mRNA translation, mRNA decay and interaction with RNA granule. RNA sequencing studies discovered that m6A modifications are highly enriched near stop codons and in 3’ untranslated regions (3’ UTR) of mRNAs. The 3’ UTR influences mRNA stability, translation efficiency, and its subcellular localization. One example of biologically important and functional methylation target 3’ UTR is given by the sex determining region y-box 2 (SOX2) mRNA which encodes for a key transcription factor and represents an oncogene. Its methylation status substantially shapes the fate of mRNA and viability of cells, respectively. However, a number of fundamental questions with respect to 3’ UTR m6A modified mRNAs and its influence on translation efficiency, kinetic and ribosome load, and RNA granules interactions are still unanswered. Utilizing 3’ UTR of SOX2 mRNA as model m6A system, a combination of cutting-edge microinjection‐based single-molecule RNA tracking methods, mRNA translation visualization techniques, and ultrahigh-resolution live cell-based single molecule fluorescence microscopy the objective of this project is to characterize the influence of 3’ UTR methylation in mRNA with respect to translation efficiency, translation kinetics, and partitioning kinetics into RNA granules (P bodies/stress granules) as well as its enrichment in RNA granules under normal and stress conditions. All this will provide unprecedented insights into understanding of m6A modification in mRNA and open a novel perspective and concept on the dynamics of the gene regulation processes collectively referred to as epitranscriptomics.