RNA修饰酶高通量筛选方法的建立及NSUN6的鉴定与功能研究

DNA、RNA和蛋白质是中心法则的基本要素，遗传信息在此之间流动。而RNA作为中心法则的重要枢纽，发挥着承上启下的作用。相比DNA和蛋白质，RNA修饰的种类最为丰富。然而，大多数RNA修饰的研究仅在rRNA和tRNA上，而许多mRNA修饰酶仍未被发现。传统寻找mRNA修饰酶的方法一种是通过分离细胞的不同组分与底物反应，进行酶活检测和质谱分析，这种方法的缺点是在体外进行，繁琐且灵敏度低；另一种则是通过生物信息学结构域比对的方法，这种方法的缺点是只能找到已有结构域的基因。因此，发展一项无偏好且高通量的筛选方法有待解决。随着CRISPR技术发展，研究者可以利用文库筛选技术寻找感兴趣的靶基因。然而，文库筛选的读出信息往往使用的是细胞殖速率的不同或者荧光信号的变化，而直接读出信息为RNA修饰还从未被探究过。本研究将sgRNA信息和RNA修饰报告基因偶联，建立了RNA修饰酶的高通量筛选方法，鉴定了新的mRNA m5C甲基转移酶NSUN6；通过对NSUN6的敲除，鉴定了NSUN6介导的位点，并揭示了其一级序列和二级结构的序列特征；通过构建Nsun6的敲除小鼠，对其生理学功能进行了系统的分析，发现Nsun6在免疫反应中具有一定的作用。

首先，本研究将CROP-seq的载体改造设计，使得sgRNA信息和RNA修饰报告基因检测偶联；利用已知的m1A甲基转移酶TRMT6/61A修饰MALAT1的m1A修饰位点作为方法的验证，成功建立将sgRNA信息和m1A修饰报告基因偶联信息共同读取的可行性；并利用已知的m5C甲基转移酶NSUN2修饰的NIBIAN2和NDUFB7的m5C位点作为验证性实验，证明了通过重亚硫酸盐处理之后，能将sgRNA信息与m5C报告基因偶联信息的可行性；针对m5C位点分析发现，存在许多非NSUN2介导的m5C修饰位点，将这些位点上下游序列克隆至本研究设计的载体上，发现这些位点仍然能够被修饰且不受到NSUN2的调控，暗示着除了唯一报道的mRNA m5C甲基转移酶NSUN2之外，还存在其他未知的修饰酶。由于该方法是基于整合gRNA和报告基因的测序技术，本研究将此方法命名为CRISPR integrated gRNA and reporter sequencing（CIGAR-seq）。

其次，通过CIGAR-seq高通量文库筛选技术，本研究使用829个RNA结合蛋白对未知的mRNA甲基转移酶进行了筛选，鉴定了全新的mRNA m5C转移酶NSUN6；通过对NSUN6的敲除，鉴定了所有NSUN6介导的m5C修饰位点，这些位点和NSUN2介导的位点构成体内所有mRNA m5C修饰位点；对NSUN6介导的m5C修饰位点序列特征进行分析，发现在一级序列上，NSUN6修饰的m5C存在明显的m5CTCCA的模体；在二级结构上，m5C修饰的位点倾向于分布在环区；但令人意外地是，NSUN6介导的m5C修饰位点对其所在的mRNA的稳定性、定位和翻译没有影响。在筛选结果中和NSUN6蛋白免疫沉淀组学实验中，都未发现明显的NSUN6辅助因子，说明NSUN6介导的m5C位点的修饰仅需要NSUN6本身实现。

最后，为了研究Nsun6的生理学功能，本研究首次构建Nsun6敲除小鼠，发现Nsun6敲除小鼠仍可以正常发育，并且纯合交配也能够生出正常的后代，这暗示着Nsun6存在着更为精细的调控方式。然后，通过对于野生型和Nsun6敲除小鼠组织的重亚硫酸盐测序，本研究总共在小肠、肝脏、脾脏、肾脏和心脏五种组织中鉴定了609个高置信度的m5C甲基化位点，其中80个是Nsun6修饰的。通过对于上述5种组织进行mRNA-seq测序，可以发现相较于其他组织，Nsun6脾脏具有一定的影响。此外，在针对于脾脏的深入研究中发现，在T细胞依赖的免疫反应中，在Nsun6敲除小鼠中，浆细胞的数量和抗体的分泌显著下降。通过结合mRNA-seq和mRNA重亚硫酸盐测序，发现Nsun6介导的m5C位点并不影响mRNA的稳定性，所以它对于免疫反应的影响是通过它的其他功能决定的。

综上，本研究主要通过建立一项高通量筛选RNA修饰酶的技术，鉴定了一个新的mRNA甲基转移酶NSUN6，并对NSUN6的分子和生理学功能进行了系统性探索，为mRNA修饰的研究提供了新的方法和基础。

DNA, RNA, and proteins are the basic elements of the central dogma, among which genetic information flows. As a hub of the central dogma, RNA plays a linking role. Compared with DNA and proteins, RNA modifications are the most abundant. However, most studies on RNA modification are limited to rRNA and tRNA, and many mRNA methyltransferases and demethylases remain undiscovered. One of the traditional methods for finding methyltransferases is to separate different components of cells and perform mass spectrometry analysis on the components that react with the substrates. The disadvantage of this method is that it is carried out in vitro, and the detection of few RNAmodified sites is not sensitive. The other method is bioinformatics prediction, the disadvantage of this method is that only genes with existing domains can be found. The high-throughput screening for these enzymes remains to be resolved. With the development of CRISPR technology, researchers can use library screening technology to search for target genes with high-throughput. However, library screening readouts are using by selective proliferation or death of cells and fluorescent reporter systems to indirectly read out the information of interest, while direct readouts for RNA modifications have never been reported. This study established a high-throughput screening method for the trans-factors of RNA modification, and identified a novel mRNA m5C methyltransferase NSUN6. Through the knockout of NSUN6, the sequence characteristics of NSUN6-dependent sites were confirmed in vivo, and explored molecular metabolism of mRNAs with NSUN6-dependent sites. Finally, by constructing Nsun6 knockout mice, its physiological functions were systematically analyzed. The CROP-seq vector was redesigned to replace the WPRE region with an RNAmodified reporter, and the endogenous sites of m1A and m5C were used to successfully clone an exogenous reporter gene that could also be modified; using the known m1A methyltranseferase and its substrate that one site in MALAT1 was used as a positive experiment, which successfully verified the feasibility of reading the sgRNA information and the m1A modification reporter gene coupling information together; and the known m5C methyltransferase NSUN2 and m5C sites of NIBIAN2 and NDUFB7 were used as positive experiments to prove the feasibility of coupling sgRNA information with m5C reporter gene information after bisulfite treatment; and found there are some NSUN2 independent m5C sites. It was found that these sites could still be modified and were not regulated by NSUN2, Since this method is based on the sequencing technology of CRISPR integrated gRNA and reporter gene, this study named it CRISPR integrated gRNA and reporter sequencing (CIGAR-seq). This study also designed a protocol for the type of RNA modification detected by immunoprecipitation for subsequent promotion. Through the high-throughput library screening technology of CIGAR-seq, this study used 829 RNA-binding proteins to screen unknown mRNA methyltransferases, and identified a novel mRNA m5C methyltransferase NSUN6; All NSUN6-dependent m5C modification sites were identified, which constitute all mRNA m5C sites with NSUN2- dependent sites; then the sequence characteristics of NSUN6-mediated m5C modification sites were analyzed, and it was found that in the primary sequence, the NSUN6-dependent sites represent motifs with obvious m5CTCCA, while in the secondary structure, they tend to be in the loop region; then the NSUN6-mediated m5C sites are explored for the mRNA molecules where they are located. Surprisingly, the NSUN6-mediated m5C modification site has no effect on the stability, localization and translation of the mRNA in which it is located, suggesting that NSUN6-mediated m5C modification may only play a role under specific conditions. Finally, the cofactors of NSUN6 were not found. In the screening results and NSUN6 protein immunoprecipitation experiments, no obvious NSUN6 cofactors were found, indicating that the NSUN6-dependent m5C site modification may only be completed by NSUN6 itself. In order to study the physiological function of Nsun6, Nsun6 knockout mice were constructed for the first time, and it was revealed that Nsun6 knockout could still develop normally, and homozygous mating could also produce normal offspring, suggesting that Nsun6 may have a more elaborate regulation. Second, through simultaneous mRNA bisulfite sequencing of wild-type and Nsun6 knockout mice, this study identified a total of 609 high-confidence m5C sites in small intestine, liver, spleen, kidney, and heart, of which are Nsun6-dependent. Through mRNA-seq of the above five tissues, it can be found that the effect of Nsun6 on the small intestine, kidney and heart is weak, while the liver and spleen have a certain effect. In addition, in an in-depth study on the spleen, for the T cell-dependent immune responses, although Nsun6 does not affect the proportion and number of germinal center B cells, it has an effect on the number of plasma cells and the secretion of antibodies. Finally, by combining mRNA-seq and mRNA bisulfite sequencing, it is suggested that Nsun6-dependent m5C sites do not affect mRNA stability, so its effect in vivo is likely to be determined by its other functions. In summary, this study identified a novel mRNA methyltransferase NSUN6 by establishing a high-throughput screening technology, and systematically explored the molecular and physiological functions of NSUN6, providing more insights into NSUN6.

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