CAREER: Chemical Biology Approaches to Study 5-Methylcytidine Derivatives in RNA and Develop CRISPR-Tet2-Based Epitranscriptomic Tool for Gene Regulation

核苷酸组成核糖核酸(RNA)，并包含细胞用来制造蛋白质的遗传信息的关键“蓝图”。通过这一奖项，化学系生命过程化学计划资助纽约州立大学奥尔巴尼分校的贾盛博士开发工具，将修饰的核苷酸高精度地整合到RNA中。这些修饰改变了RNA的结构和功能。这些变化帮助我们理解了RNA在众多生物过程中的作用。这项研究的重点是确定含有修饰核苷酸的RNA的三维结构，了解这些变化是如何影响RNA的反应活性的，然后研究核苷酸在基因表达中的作用。盛博士使新的天然核苷酸及其3D结构可供广泛的研究人员使用。根据该项目开发的策略可能适用于针对不同RNA修饰的其他酶，从而产生一种新的一般RNA编辑方法，因此，这项研究被认为是在理解生命规则倡议的范围内。这项研究的研究方法和成果被整合到一门新的课程《核酸化学》中。盛博士是“化学世界”项目的带头人，该项目帮助化学专业的新生顺利过渡到大学。盛博士还在他的实验室为当地的K-12学生组织了一年一度的“RNA日”活动，为他们提供实验演示和演讲，以唤醒他们内心的科学家。最后，课题组招募学生和老师参与“高中大学”计划的暑期研究。这一奖项使研究成为可能，研究重点是RNA中化学修饰的胞嘧啶的相对较新的表观遗传学意义。合成的5-甲基胞嘧啶(5mC)及其氧化变异体(5xrC)被系统地掺入RNA中，以确定它们对RNA的稳定性以及局部和全局构象的影响。此外，对5xrC修饰的RNA的研究揭示了它们对酶识别的影响，以及加氧酶Tet家族对5mC的酶催化氧化机制。最后，以簇状规则间隔短回文重复序列(CRISPR)为靶点的序列与Tet加氧酶融合，创建一种工具，选择性地氧化天然RNA中的5mC。这个工具被用来确定5xrC核苷酸如何影响将细胞mRNA翻译成蛋白质的效率。盛博士所获得的知识和开发的新的通用方法可能会对其他研究真核生物中信使RNA的表观转录修饰和基因表达转录后调控的科学家有所裨益。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Nucleotides make up ribonucleic acids (RNAs) and contain the crucial "blueprint" of genetic information that is used by cells to make proteins. With this award, the Chemistry of Life Processes Program in the Division of Chemistry is funding Dr. Jia Sheng, from the State University of New York at Albany, to develop tools to incorporate modified nucleotides into RNA with high precision. These modifications change the RNA structure and function. These changes help us understand the role of RNA in numerous biological processes. The research focuses on the determining the three-dimensional structure of RNAs containing the modified nucleotides, understanding how these changes affect the reactivity of the RNA and then studying the study of the nucleotides' role in gene expression. The Dr. Sheng makes the new natural nucleotides and their 3D structures available to a wide range of researchers. The strategies developed under this project may be applicable to other enzymes targeting different RNA modifications, leading to a new general approach for RNA editing and thus, this research is considered to be within the Understanding the Rules of Life initiative. The research methodologies and outcomes of the study are integrated into a new course "Nucleic Acid Chemistry". Dr. Sheng is a leader in the "World of Chemistry" program which helps freshmen chemistry majors transition to college successfully. Dr. Sheng also organizes the annual "RNA Day" in his lab for local K-12 students offering experimental demonstrations and presentations to awaken their inner scientist. Finally, the research group recruits students and teachers to participate in summer research through the "University in the High School" program.The research studies made possible by this award focus on the relatively new epigenetic significance of chemically-modified cytosines in RNA. Synthetic 5-methylcytosine (5mC) and its oxidized variants (5xrC) are systematically incorporated into RNAs to determine their impact on the stability and local and global conformations of the RNAs. In addition, studies with 5xrC-modified RNAs reveal their effects on enzyme recognition and the mechanism of enzymatic oxidation of 5mC by the ten-eleven translocation (TET) family of oxygenases. Finally, the sequence targeting the clustered regularly interspaced short palindromic repeats (CRISPR) enzyme is fused with a TET oxygenase to create a tool that site-selectively oxidizes 5mC in natural RNAs. This tool is used to determine how 5xrC nucleotides affect the efficiency of translating cellular mRNAs into proteins. The knowledge gained and new general approaches developed by Dr. Sheng may benefit other scientists who study epitranscriptomic modifications of messenger RNA and posttranscriptional regulation of gene expression in eukaryotes.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.