ArticleCell & bioscience2026
lncRNA-operated stem cell fate control: RPS3 mediates the in situ Gm16751-Zhx3 interactome to program spermatogonial stem cell differentiation.
Article in Cell & bioscience, 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
backgroundLong 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.
methodsThe 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.
resultsWe 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. Furthermore, mass spectrometry analysis revealed that RPS3 (Ribosomal Protein S3) serves as a critical RBP that bridges the interaction between Gm16751 and Zhx3, ultimately stabilizing Zhx3 mRNA and modulating SSC differentiation.
conclusionsOur study identified a novel lncRNA-mRNA interaction pair, Gm16751/Zhx3, which is mediated by RPS3 and facilitates the differentiation of SSC. This discovery provides important mechanistic insights into the regulation of male germ cell development and offers a new perspective for understanding molecular controls in spermatogenesis.
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