(324) RNA m⁶A Dependent Alternative Splicing of Ankyrin Repeat Domain 46 Accelerates Ovarian Aging

Abstract Authors: Lihua Zeng^{1, 2}; Jiani Wan¹; Yunling Zheng²; Lijun Ruan³; Huijuan Peng²; Manish Kumar¹; Ling Zhu²; Jing Liu¹

1. Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China
2. Guangzhou University of Chinese Medicine, Guangzhou, China
3. The Fifth Affiliated Hospital of Southern Medical University, Guangzhou, China

Abstract Text:

Ovarian aging is the onset of the systemic aging. And it would lead to infertility and menopause. According to the hallmarks of ovarian aging, epigenetic alterations play an important role. RNA m⁶A is a conserved and common post-transcriptional modification, and it has been recognized to be strongly related to ovarian aging. However, the underlying mechanism of RNA m⁶A modification on ovarian aging remains unknown. In this study, firstly, ovaries and blood samples from both young (20-35 years old) and old women (40-48 years) were collected after approvement of Ethics Committees and acquiring informed consent. Aging phenotypes including serum anti-mullerian hormone (AMH) levels and expression of p16 in ovaries were tested with enzyme-linked immunosorbent assay (ELISA) and immunofluorescence (IF). Then RNA m⁶A modification including m⁶A levels and expressions of m⁶A related enzymes were tested with ELISA, western blot and immunohistochemistry. The specific differential m⁶A modified genes were analyzed with MeRIP-seq. Secondly, human granulosa cell line KGN was chosen for in vitro experimental studies. After sliencing the differential m⁶A modified enzyme with siRNA, downstream target genes were analyzed with RNA-seq. Combining with the MeRIP-seq data, overlapped genes were verified with MeRIP-qPCR. Then splicing variant of the target gene was tested with fluorescence in situ hybridization (FISH). To further test the functions of target gene, aging phenotypes including senescence β-galactosidase (SA-β-Gal) levels and p16 expression were tested with SA-β-Gal staining and IF after overexpressing the splicing variant. Additionally, migration function was also tested after overexpressing the splicing variant. For clinical samples, the old group showed significant lower AMH levels (P< 0.001) and higher p16 expressions (P< 0.05) comparing to the young group. In RNA m⁶A modification, the old group showed higher RNA m⁶A level (P< 0.05), which was mainly attributed to downregulated demethyltransferase alpha-ketoglutarate-dependent dioxygenase AlkB homolog 5 (ALKBH5) especially expressed in granulosa cells (P< 0.05). Combing analyzing the MeRIP-seq data from human ovaries and RNA-seq data from si-ALKBH5 KGN, 10 overlapped genes were found to be candidate target genes modified by ALKBH5. After sliencing and overexpressing ALKBH5, those 10 candidate target genes were further verified with MeRIP-qPCR, and ankyrin repeat domain 46 (ANKRD46) was verified to be the final target gene. Apart from regulating the expression of RNA, RNA m⁶A could also promote the production of alternative splicing variants. Alternative splicing analysis showed that m⁶A modification caused by downregulated ALKBH5 could lead to the upregulation of splicing variant 4 of ANKRD46 through alternative 3’ splice site. And FISH also indicated the upregulated splicing variant 4 of ANKRD46 (P< 0.05) after sliencing ALKBH5. Besides, functional experiments showed that overexpressing ANKRD46 variant 4 increased β-galactosidase (SA-β-Gal) levels and p16 expressions (P< 0.05), which indicated ANKRD46 variant 4 accelerated the granulosa cells aging. Additionally, overexpressing ANKRD46 variant 4 could also damage the migration function of KGN, which may indicate the negative effect of ANKRD46 variant 4 on cumulus-oocyte complex expansion. In conclusion, our study revealed that ANKRD46 splicing variant 4 produced by RNA m⁶A dependent alternative splicing could accelerate ovarian aging, which may be related to failure in cumulus-oocyte complex expansion. This work was supported by National Natural Science Foundation of China (No.82174418, 32370791) and Postdoctoral Fellowship Program (Grade C) of China Postdoctoral Science Foundation (No.GZC20232689).