Abstract

Long non-coding RNAs (lncRNAs) play pivotal regulatory roles in mammalian male gametogenesis. Advances in high-throughput technologies have demonstrated that lncRNAs orchestrate robust, flexible, and context-specific gene regulatory networks through dynamic interactions with proteins, DNA, and RNA, modulating transcriptional and post-transcriptional processes. However, the mechanistic role of in situ lncRNA-mRNA interactions in spermatogonial stem cell (SSC) differentiation remains poorly understood. The differentially expressed transcripts, including lncRNA, mRNA and circRNA, were systematically identified through long RNA sequencing (LongRNA-seq). RNA-RNA in situ interactions were subsequently mapped using RNA in situ conformation sequencing (RIC-seq) technology. The Gm16751–Zhx3 interaction was validated using single-molecule fluorescence in situ hybridization (smFISH), coupled with RNA antisense purification followed by quantitative PCR (RAP-qPCR), and dual-luciferase reporter assay. Its functional impact on SSC differentiation was assessed via EdU staining, apoptosis assay, mRNA stability assay, RAP-qPCR, respectively. Liquid chromatography-tandem mass spectrometry (LC-MS) and RNA immunoprecipitation-qPCR (RIP-qPCR) were employed to screen and identify the RNA-binding protein (RBP) interacting with Gm16751 and Zhx3. Crosslinking immunoprecipitation followed by qPCR (CLIP-qPCR) further identified specific binding sequences. The regulation of RBP on the differentiation of SSC was verified using RAP-qPCR, RIP-qPCR, puromycin incorporation assay, and RNA stability assay. We employed RIC-seq and LongRNA-seq to systematically identify a functional interaction between lncRNA Gm16751 and Zhx3 mRNA during SSC differentiation, which was further visualized via a Circos plot. Mechanistically, we demonstrated that Gm16751 enhances the mRNA stability of Zhx3, thereby promoting SSC differentiation.