24-Epibrassinolide drives female germline stem cell development via NMT1-mediated in situ interaction between Gm26624 and Abcg2

Recent studies indicate that approximately 15% of couples worldwide are affected by infertility. The discovery of female germline stem cells (FGSCs) offers a promising new therapeutic strategy for infertility treatment.The objective of this study is to elucidate the regulatory role of epibrassinolide in the development of FGSCs and its underlying mechanism. We developed an in vitro FGSC culture system treated with 24-epibrassinolide (EBR, E1641, Sigma), a bioactive brassinosteroid. Proliferation was quantified via CCK-8 assays and EdU incorporation; apoptotic levels of cells following drug treatment were assessed by flow cytometry and TUNEL assay; differentiation status was assessed by western blotting, immunofluorescence, and qRT-PCR. For exploring mechanisms，we performed long-read RNA-seq, RNA in situ conformation sequencing (RIC-seq), tagged RNA affinity purification, LC-MS/MS proteomics, single-molecule RNA FISH, and RNA immunoprecipitation. EBR significantly enhanced FGSC development by coordinately regulating: (1) meiotic progression acceleration and (2) apoptotic pathway suppression. Integrative RIC-seq/longRNA-seq analysis revealed EBR reinforces spatial interactions between lncRNA Gm26624 and transporter gene Abcg2. Mechanistically, N-myristoyltransferase 1 (NMT1) functions as an RNA-binding scaffold facilitating this RNA-RNA interaction. Functional validation established the EBR-induced Gm26624-Abcg2 axis as both necessary and sufficient for meiotic entry initiation. We identify a phytohormonal signaling pathway orchestrating FGSC fate determination through an NMT1-mediated RNA interactome. These findings elucidate fundamental germline stem cell regulation principles and provide proof-of-concept for plant-derived compounds as promising reproductive therapeutics. Working model: 24-Epibrassinolide drives female germline stem cell development via NMT1-mediated in situ interaction between Gm26624 and Abcg2 • First evidence that EBR promotes FGSC development. • Gm26624–Abcg2 interaction identified as a key regulator via LongRNA-seq and RIC-seq. • EBR acts through NMT1-mediated Gm26624–Abcg2 interaction.