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<p>Profiling the protein interactome of specific RNA loci aids in understanding the molecular mechanisms of regulatory RNA. However, current RNA-centric methods have sufficient space for improvement in terms of efficiency and biocompatibility. Here we developed TurboID-assisted proximity labeling of targeted RNA-interacting proteins (TAPRIP), in which the proximity labeling enzyme miniTurbo is attached to an RNA-targeting element, CIRTS3, to label the targeted RNA-interacting proteins, which are then analyzed by mass spectrometry. We profiled the interactome of mCherry circular RNA (circRNA) and found that HNRNPK modulates the expression of mCherry circRNA and other endogenous circRNAs by binding their flanking introns. Targeting the <i>BCL2</i> RNA G-quadruplex structure, we found that RBM12 promotes <i>BCL2</i> translation by binding RNA G-quadruplexes and recruiting ribosomes. <i>RBM12</i> knockdown markedly reduced proliferation and clonogenicity in MOLM-13, MV4-11 and THP-1 acute myeloid leukemia cells. These findings established the foundation for a versatile technique for analyzing the protein interactome of specific RNA sequences in live cells.</p><p></p>

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In situ proximity labeling of proteins associated with circRNA and RNA G-quadruplexes

  • 期刊论文

  • 发表日期：

    2025年8月7日

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  • Jing Mo,
  • Zonggui Chen,
  • Manman Cui,
  • Xin Fang,
  • Ruihan Li,
  • Shanshan Qin,
  • Zhenlin Zhou,
  • Mei Sun,
  • Jinjun Wu,
  • Chuan He,
  • Fang Wang,
  • Haojian Zhang,
  • Xiang Zhou,
  • Xiaocheng Weng

摘要

Profiling the protein interactome of specific RNA loci aids in understanding the molecular mechanisms of regulatory RNA. However, current RNA-centric methods have sufficient space for improvement in terms of efficiency and biocompatibility. Here we developed TurboID-assisted proximity labeling of targeted RNA-interacting proteins (TAPRIP), in which the proximity labeling enzyme miniTurbo is attached to an RNA-targeting element, CIRTS3, to label the targeted RNA-interacting proteins, which are then analyzed by mass spectrometry. We profiled the interactome of mCherry circular RNA (circRNA) and found that HNRNPK modulates the expression of mCherry circRNA and other endogenous circRNAs by binding their flanking introns. Targeting the BCL2 RNA G-quadruplex structure, we found that RBM12 promotes BCL2 translation by binding RNA G-quadruplexes and recruiting ribosomes. RBM12 knockdown markedly reduced proliferation and clonogenicity in MOLM-13, MV4-11 and THP-1 acute myeloid leukemia cells. These findings established the foundation for a versatile technique for analyzing the protein interactome of specific RNA sequences in live cells.