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Updated: Apr 30, 2026

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3'UTR-reporter Constructs and a microRNA Mimic in Adherent Cells
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TRIM65 regulates microRNA activity by ubiquitination of TNRC6

Shitao Li1, Lingyan Wang2, Bishi Fu2

  • 1Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115 shitao_li@hms.harvard.edu dorf@hms.harvard.edu.

Proceedings of the National Academy of Sciences of the United States of America
|April 30, 2014
PubMed

Insights

Tripartite motif 65 (TRIM65) protein was discovered to regulate microRNA (miRNA) activity by targeting TNRC6 proteins for degradation, thereby relieving miRNA-mediated gene silencing.

Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • MicroRNAs (miRNAs) are crucial regulators of gene expression, influencing cellular processes like proliferation and apoptosis.
  • Identifying novel regulators of miRNA pathways is essential for understanding gene regulation.

Purpose of the Study:

  • To systematically identify novel regulators of microRNA activity in human cells.
  • To elucidate the mechanism by which Tripartite motif 65 (TRIM65) influences miRNA function.

Main Methods:

  • Proteomic analysis of the miRNA pathway interactome.
  • Reporter assays to measure miRNA activity.
  • Co-localization studies and ubiquitination assays.
  • Overexpression and knockdown experiments.

Main Results:

  • A proteomic screen identified 66 potential miRNA pathway interactors, with six genes regulating let-7a miRNA activity.
  • TRIM65 was identified as a repressor of miRNA activity.
  • TRIM65 interacts with and ubiquitinates TNRC6 proteins, leading to their degradation.
  • TRIM65 overexpression relieved miRNA-driven mRNA suppression.

Conclusions:

  • TRIM65 acts as an E3 ubiquitin ligase for TNRC6 proteins, promoting their degradation.
  • TRIM65 antagonizes miRNA activity by targeting TNRC6, a key component of the miRNA-induced silencing complex.
  • This finding reveals a novel mechanism for regulating miRNA function and gene expression.
Keywords:
GW proteinsRNA-induced silencing complexprotein interaction networks

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