The m6A demethylase ALKBH5 controls trophoblast invasion at the maternal-fetal interface by regulating the stability of <i>CYR61</i> mRNA

Abstract

N⁶-Methyladenosine (m⁶A) is the most prevalent internal modification in mammalian mRNAs. Although m⁶A is important in many biological processes, its roles in the placenta are unclear. <b>Methods</b>: Levels of global mRNA m⁶A methylation and ALKBH5 expression in recurrent miscarriage (RM) patients were determined using quantitative reverse transcription-PCR (qRT-PCR), m⁶A RNA methylation quantification, and immunohistochemical methods. Using ALKBH5 overexpression and knockdown methods, we determined the role of ALKBH5 in trophoblast invasion at the maternal interface through trophoblasts and an extravillous explant culture experiments. Furthermore, the regulation of CYR61 by ALKBH5 was explored by RNA-sequencing coupled with methylated RNA immunoprecipitation. <b>Results</b>: We found that the level of global mRNA m⁶A methylation was significantly decreased in placental villous tissue from RM patients, while ALKBH5 expression was specifically unregulated. Furthermore, we demonstrated that ALKBH5 knockdown in human trophoblast promoted trophoblast invasion. Conversely, overexpression of ALKBH5 inhibited cell invasion. ALKBH5 knockdown promoted trophoblast invasion in villous explant culture experiments, while overexpression of ALKBH5 repressed these effects. Furthermore, we clarified that ALKBH5 inhibited trophoblast invasion by regulating <i>CYR61</i> mRNA stability, and this RNA regulation is m⁶A dependent. Mechanistic analyses showed that decreased <i>ALKBH5</i> in trophoblast increased the half-life of <i>CYR61</i> mRNA and promoted steady-state <i>CYR61</i> mRNA expression levels. <b>Conclusions</b>: We elucidated the functional roles of ALKBH5 and mRNA m⁶A methylation in trophoblast and identified a novel RNA regulatory mechanism, providing a basis for further exploration of broad RNA epigenetic regulatory patterns in RM diseases.