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A Reporter Assay to Analyze Intronic microRNA Maturation in Mammalian Cells
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Negative regulation of miRNA sorting into EVs is mediated by the capacity of RBP PCBP2 to impair the

Francesco Marocco1, Sabrina Garbo1, Claudia Montaldo2

  • 1Istituto Pasteur Italia-Fondazione Cenci Bolognetti, Department of Molecular Medicine, Department of Excellence 2023-2027, Sapienza University of Rome, Rome, Italy.

Elife
|July 2, 2025
PubMed

Insights

Poly(rC) binding protein 2 (PCBP2) inhibits microRNA (miRNA) loading into extracellular vesicles (EVs). PCBP2 acts as a dominant inhibitor of SYNCRIP, a known miRNA EV-loader, in hepatocytes.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biochemistry

Background:

  • Extracellular vesicles (EVs) mediate intercellular communication via microRNA (miRNA) transfer.
  • Mechanisms controlling selective miRNA loading into EVs are not fully understood.
  • Intracellular retention motifs for miRNAs exist, but their interacting proteins are unknown.

Purpose of the Study:

  • To identify proteins interacting with intracellular miRNA retention motifs.
  • To elucidate the role of these interactions in miRNA loading into EVs.
  • To understand the interplay between PCBP2, SYNCRIP, and miRNA compartmentalization.

Main Methods:

  • Cross-linked immunoprecipitation coupled to RNA pull-down (CLIP) and proteomic analysis.
  • Mutagenesis analysis to confirm binding specificity.
  • SYNCRIP knockdown experiments and Electrophoretic Mobility Shift Assays (EMSA).

Main Results:

  • PCBP2 directly binds to miRNAs containing a specific intracellular retention motif.
  • PCBP2 binding to miRNAs is dependent on SYNCRIP.
  • PCBP2 and SYNCRIP can bind to miRNAs simultaneously.
  • PCBP2 knockdown leads to increased loading of intracellular miRNAs into EVs.

Conclusions:

  • Multiple protein-miRNA interactions regulate miRNA compartmentalization and EV loading.
  • PCBP2 acts as a dominant inhibitor of SYNCRIP's function in miRNA loading.
  • PCBP2 plays a key role in controlling miRNA release via extracellular vesicles in hepatocytes.
Keywords:
RNA-binding proteinscell biologyextracellular vesiclesmiRNAmouse

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