m6A修饰调控PLK1蛋白表达及其对有丝分裂的影响

m6A modification regulates PLK1 expression and mitosis

摘要:

N⁶-甲基腺苷(N⁶-methyladenosine,m⁶A)修饰在细胞周期调控中起着关键作用。为了研究m⁶A调控有丝分裂的机制，本研究首先进行了液相色谱-串联质谱和m⁶A斑点印迹试验，发现细胞内总m⁶A修饰水平在有丝分裂期间升高。通过免疫荧光技术，发现沉默甲基转移酶样蛋白3(methyltransferase-like 3,METTL3)和甲基转移酶样蛋白14(methyltransferase-like 14,METTL14)会导致有丝分裂延迟、纺锤体组装异常和染色体分离缺陷。之后对HeLa细胞中转录组范围内的m⁶A靶点进行分析，发现polo样蛋白激酶1(polo-like kinase 1,PLK1)是调节有丝分裂的关键m⁶A修饰基因。最后，采用免疫印记和RNA pulldown试验，发现PLK1 mRNA的m⁶A修饰被YTH结构域家族蛋白1(YTH N⁶-methyladenosine RNA binding protein 1,YTHDF1)结合并抑制PLK1的蛋白表达，从而调控细胞周期。PLK1 mRNA的去甲基化提高了PLK1蛋白的表达，导致有丝分裂异常。本研究揭示了m⁶A在有丝分裂调控中的关键作用及其作为癌症等疾病的治疗靶点的潜力。

Abstract:

N⁶-methyladenosine (m⁶A) modification plays a critical role in cell cycle regulation, while the mechanism of m⁶A in regulating mitosis remains underexplored. Here, we found that the total m⁶A modification level in cells increased during mitosis by the liquid chromatography- mass spectrometry/mass spectrometry and m⁶A dot blot assays. Silencing methyltransferase-like 3 (METTL3) or METTL14 results in delayed mitosis, abnormal spindle assembly, and chromosome segregation defects by the immunofluorescence. By analyzing transcriptome-wide m⁶A targets in HeLa cells, we identified polo-like kinase 1 (PLK1) as a key gene modified by m⁶A in regulating mitosis. Specifically, through immunoblotting and RNA pulldown, m⁶A modification inhibits PLK1 translation via YTH N⁶-methyladenosine RNA binding protein 1, thus mediating cell cycle homeostasis. Demethylation of PLK1 mRNA leads to significant mitotic abnormalities. These findings highlight the critical role of m⁶A in regulating mitosis and the potential of m⁶A as a therapeutic target in proliferative diseases such as cancer.