甲基转移酶NSUN2能够介导多种类型RNA的m⁵C甲基化修饰，影响RNA的稳定性及转录和翻译过程，参与调控细胞的生长、增殖和分化等重要生物学活动，在生物的个体发育、衰老、自身免疫以及肿瘤的发生发展过程中发挥重要的作用。

Autotaxin（ATX）是一种具有溶血磷脂酶D（lysoPLD）活性的分泌型糖蛋白，能够催化溶血磷脂酰胆碱（LPC）水解生成溶血磷脂酸（LPA），是体内催化LPA生成的关键酶。LPA是一种具有生物活性的脂质分子，通过特异性结合细胞膜上LPA受体参与多种生理和病理过程的调控。ATX-LPA信号轴在个体发育、免疫调控、心血管疾病、肥胖和肥胖相关疾病以及肿瘤的发生和发展（特别是在肿瘤细胞的迁移）过程中发挥作用。已有的对ATX表达调控机制的研究主要集中在转录水平，对ATX的转录后表达调控机制，特别是RNA甲基化修饰对ATX的表达调控尚缺乏了解，本论文着重探讨mRNA甲基化修饰在转录后水平对ATX表达的调控机制及其生物学意义。

我们发现ATX mRNA上存在m⁵C而没有m⁶A甲基化修饰，mRNA m⁵C甲基转移酶NSUN2能够结合ATX mRNA，并在体内和体外均能对ATX mRNA进行甲基化修饰。ATX mRNA 的m⁵C修饰发生于3'UTR，修饰位点主要在2756位胞嘧啶。在肿瘤细胞中沉默NSUN2显著降低ATX的表达，过表达NSUN2则显著升高ATX的表达，但ATX的mRNA表达水平均不受影响，表明NSUN2在转录后水平正调控ATX的表达。通过构建报告基因质粒，我们发现NSUN2能正向调控携带野生型ATX 3'UTR的报告基因（pGL3-3'UTR）活性，但不影响携带5'UTR、CDS和甲基化位点突变的3'UTR（pGL3-3'UTR-M）的报告基因活性。体外翻译系统中，NSUN2介导的ATX 3'UTR甲基化修饰能提高pGL3-3'UTR报告基因转录本的翻译效率，但对pGL3-3'UTR-M报告基因转录本的翻译效率没有作用。而且，沉默NSUN2可以降低细胞中ATX mRNA在多聚核糖体中的分布。此外，NSUN2介导的m⁵C修饰能促进ATX mRNA的出核运输，该过程依赖于mRNA m⁵C结合蛋白ALYREF。在人神经胶质瘤细胞系U87中沉默NSUN2可以显著抑制肿瘤细胞的迁移能力，而在细胞培养基中加入LPA能使沉默NSUN2的细胞的迁移能力得到明显的回复。

我们的研究发现，NSUN2不仅参与肿瘤细胞中ATX 的表达调控，而且在小鼠脂肪细胞ATX的表达调控中发挥作用。此外，与野生型小鼠相比，NSUN2杂合敲除小鼠脑中ATX蛋白水平显著下降，但ATX mRNA水平没有显著差异，而且在NSUN2纯合敲除小鼠脑中ATX蛋白表达量比杂合敲除小鼠的下降得更多。

        上述结果表明，NSUN2能催化ATX mRNA 3'UTR发生m⁵C甲基化修饰，促进ATX mRNA出核运输并提高其翻译效率，从而促进ATX蛋白的表达，NSUN2-ATX-LPA轴对肿瘤细胞的迁移具有调控作用。此外，NSUN2对ATX的表达调控也发生在脂肪细胞和脑组织，其生物学意义还有待进一步阐释。本研究从NSUN2介导的mRNA m⁵C甲基化修饰的角度，提出了一个新的ATX的转录后表达调控机制，有助于全面认识NSUN2和ATX的生物学功能和作用机制。

The methyltransferase NSUN2 can mediate m⁵C methylation modification on various types of RNA, to affect the stability of RNA and the process of transcription and translation, and participate in the regulation of cell growth, proliferation, differentiation and other important biological activities, playing a vital part in ontogenesis, aging, autoimmunity and the occurrence and development of tumors.

Autotaxin (ATX) is a secreted glycoprotein with lysophospholipase D (lysoPLD) activity, and functions as the key enzyme to convert lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA) in vivo. LPA is a bioactive lysophospholipid, which is involved in various physiological and pathological processes by specifically binding to LPA receptors on the cell membrane. The ATX-LPA axis plays a role in the process of ontogeny, immune regulation, cardiovascular disease, obesity and obesity-related diseases, as well as the occurrence and development of tumors (especially in tumor cell migration). The existing researches on the regulation mechanisms of ATX expression mainly focus on the transcription level. The post-transcriptional regulation mechanisms of ATX expression, especially by RNA methylation modification, are still lacking in understanding. This paper focuses on the regulatory mechanism of mRNA methylation modification on ATX expression at the post-transcriptional level and its biological significance.

We found that m⁵C, but not m⁶A, was present on ATX mRNA. The mRNA m⁵C methyltransferase NSUN2 could bind to ATX mRNA and methylate it both in vivo and in vitro. The m⁵C modification occurred at the 3'-untranslated region (3'UTR) of ATX mRNA and mainly at cytosine 2756. Silencing NSUN2 in tumor cells significantly decreased the protein levels of ATX, while the overexpression of NSUN2 notably increased the expression of ATX, but the mRNA levels of ATX were not affected, suggesting that NSUN2 positively regulated ATX expression at the post-transcriptional level. Using reporter gene assays, we found that NSUN2 could positively regulate the activity of pGL3-derived reporter bearing ATX 3'UTR (pGL3-3'UTR), but not that bearing ATX 5'UTR, CDS or 3'UTR mutating at C2756 (pGL3-3'UTR-M). In an in vitro translation system, NSUN2-mediated ATX 3'UTR methylation increased the translation efficiency of chimeric transcripts transcribed from pGL3-3'UTR reporter, but not that of chimeric transcripts transcribed from pGL3-3'UTR-M reporter. Moreover, knockdown of NSUN2 could reduce the presence of ATX mRNA in the polysome. In addition, NSUN2-mediated m⁵C modification promoted the nuclear export of ATX mRNA, which was dependent on the mRNA m⁵C binding protein ALYREF. Silencing NSUN2 could significantly suppress the migration of human glioma cells U87, while the addition of LPA to the cell culture medium could remarkably rescue the migration ability of the cells with silenced NSUN2.

Our research found that the regulation of ATX expression by NSUN2 not only existed in tumor cells, but also played a part in mouse adipocytes. In addition, compared with wild-type mice, ATX protein levels in the brain of NSUN2 heterozygous knockout mice were markedly reduced, however, there was no significant difference in ATX mRNA levels. Besides, compared with NSUN2 heterozygous mice, ATX protein levels further decreased in the brain of NSUN2 homozygous knockout mice.

        The above results indicate that NSUN2 can promote the nuclear transport of ATX mRNA and improve its translation efficiency by catalyzing the m⁵C methylation modification on ATX mRNA 3'UTR, thereby increasing the expression of ATX. The NSUN2-ATX-LPA axis has a regulatory effect on the migration of tumor cells. In addition, the regulation of ATX expression by NSUN2 also occurs in adipocytes and brain tissue, and the related biological functions remain to be further elucidated. From the perspective of NSUN2-mediated mRNA m⁵C methylation modification, this study presents a new post-transcriptional regulation mechanism of ATX expression, which is conducive to the comprehensively understanding of the biological functions and mechanisms of NSUN2 and ATX.