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Background <p>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.</p> Methods <p>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 <i>Gm16751</i>–<i>Zhx3</i> 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 <i>Gm16751</i> and <i>Zhx3</i>. 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.</p> Results <p>We employed RIC-seq and LongRNA-seq to systematically identify a functional interaction between lncRNA <i>Gm16751</i> and <i>Zhx3</i> mRNA during SSC differentiation, which was further visualized via a Circos plot. Mechanistically, we demonstrated that <i>Gm16751</i> enhances the mRNA stability of <i>Zhx3</i>, thereby promoting SSC differentiation. Furthermore, mass spectrometry analysis revealed that RPS3 (Ribosomal Protein S3) serves as a critical RBP that bridges the interaction between <i>Gm16751</i> and <i>Zhx3</i>, ultimately stabilizing <i>Zhx3</i> mRNA and modulating SSC differentiation.</p> Conclusions <p>Our study identified a novel lncRNA-mRNA interaction pair, <i>Gm16751</i>/<i>Zhx3</i>, 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.</p> Graphical abstract <p></p>

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lncRNA-operated stem cell fate control: RPS3 mediates the in situ Gm16751–Zhx3 interactome to program spermatogonial stem cell differentiation

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年6月2日

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  • Weijun Gao,
  • Bo Xu,
  • Wenzhi Ma,
  • Xiaojin He,
  • Ji Wu

摘要

Background

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.

Methods

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.

Results

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. 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.

Conclusions

Our 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.

Graphical abstract