18S ribosomal RNA methyltransferase METTL5-mediated CDX2 translation regulates porcine early embryo development

doi:10.1016/j.jia.2023.10.013

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XU Teng-teng^*, ZHANG Meng-ya^*, LIU Qiu-chen^*, WANG Xin^*, LUO Peng-fei, LIU Tong, YAN Ye-lian, ZHOU Na-ru, MA Yang-yang, YU Tong, LI Yun-sheng, CAO Zu-bing^#, ZHANG Yun-hai^#

Anhui Province Key Laboratory of Local Livestock and Poultry, Genetical Resource Conservation and Breeding, College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, P.R.China

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摘要

目的：探究18S核糖体RNA m6A甲基转移酶METTL5在早期胚胎发育及细胞谱系分化中的功能及调控机制。方法：利用荧光定量PCR（qPCR）、免疫荧光染色（IF）及蛋白免疫印迹（WB）分析METTL5在猪早期胚胎中的动态表达，利用显微操作技术分别在孤雌激活（PA）和体外受精（IVF）胚胎中敲低和过表达METTL5，观察对猪早期胚胎发育效率的影响。收集对照组、METTL5敲低或过表达胚胎，利用qPCR、IF及WB分析METTL5缺失或过表达对细胞谱系分化标志物表达与定位的影响，利用OP-Puro试剂盒检测METTL5缺失及过表达对整体翻译效率的影响。结果：METTL5在猪早期胚胎中持续表达，METTL5敲低不影响卵裂率，但显著降低了猪早期胚胎发育效率（PA囊胚率：对照组 vs METTL5敲低=58.8±1.92% vs 38.34±1.28%，P<0.05；IVF囊胚率：对照组 vs METTL5敲低=23.27±3.08% vs 9.69±3.08%，P<0.05）。此外，METTL5过表达也显著降低了囊胚发育效率（对照组 vs METTL5过表达=59.3±3.9% vs 37.5±2.6%，P<0.05）。METTL5敲低不影响mRNA m6A水平，也不影响紧密连接的形成，但METTL5敲低降低了CDX2蛋白的表达水平，并升高了NANOG和OCT4 mRNA水平。METTL5敲低及过表达分别降低及升高了整体mRNA的翻译水平。值得注意的是，CDX2过表达可以挽救METTL5缺失胚胎的发育效率，并部分挽救了整体mRNA的翻译效率。结论：METTL5对猪囊胚形成及细胞谱系分化至关重要，METTL5通过调节CDX2的翻译进而调控猪早期胚胎发育。创新性：本研究率先揭示了METTL5在猪早期胚胎发育中的功能及调控机制，为加速畜禽繁殖及早期胚胎发育效率的提高提供科学依据。

Abstract

N6-methyladenosine (m6A) plays a key role in mammalian early embryonic development and cell lineage differentiation. However, the role and mechanisms of 18S ribosomal RNA (rRNA) m6A methyltransferase METTL5 in early embryonic development remain unclear. Here, we found that 18S rRNA m6A methyltransferase METTL5 plays an important role in porcine early embryonic development. METTL5 knockdown and overexpression significantly reduced the developmental efficiency of porcine early embryos and impaired cell lineage allocation. METTL5 knockdown apparently decreased the global translation efficiency in blastocyst, while METTL5 overexpression increased the global translation efficiency. Furthermore, METTL5 knockdown did not affect the abundance of CDX2 mRNA, but resulted in a significant reduction in CDX2 protein levels. Moreover, the low developmental efficiency and abnormal lineage distribution of METTL5 knockdown embryos could be rescued by CDX2 overexpression. Collectively, our results demonstrated that 18S rRNA methyltransferase METTL5 regulates porcine early embryonic development via modulating the translation of CDX2.

Keywords: pig rRNA m6A CDX2 mRNA translation lineage segregation

Online: 19 October 2023 Accepted:

Fund: This work was supported by grants from the Sub-project of National Key Research and Development Program of China (2021YFA0805905-1), the Special Fund for Anhui Agriculture Research System (AHCYJSTX-04), the Joint Research Project on the Anhui Local Pigs Breeding and Utilization (340000211260001000431), the Open Project of key Laboratory of Embryo Development and Reproductive Regulation Anhui Province (FSKFKT004), and Major special science and technology project of Anhui Province (202103a06020013).

About author: #Correspondence ZHANG Yun-hai, E-mail: yunhaizhang@ahau.edu.cn; CAO Zu-bing Cao, E-mail: zubingcao@ahau.edu.cn * These authors contributed equally to this study.

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XU Teng-teng, ZHANG Meng-ya, LIU Qiu-chen, WANG Xin, LUO Peng-fei, LIU Tong, YAN Ye-lian, ZHOU Na-ru, MA Yang-yang, YU Tong, LI Yun-sheng, CAO Zu-bing, ZHANG Yun-hai. 2023. 18S ribosomal RNA methyltransferase METTL5-mediated CDX2 translation regulates porcine early embryo development. Journal of Integrative Agriculture, Doi:10.1016/j.jia.2023.10.013

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