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Updated: Dec 16, 2025
A Reporter Assay to Analyze Intronic microRNA Maturation in Mammalian Cells
Published on: June 16, 2022
The human box C/D snoRNA U3 is a miRNA source and miR-U3 regulates expression of sortin nexin 27
Nicolas Lemus-Diaz1,2, Rafael Rinaldi Ferreira2, Katherine E Bohnsack1
1Department of Molecular Biology, University Medical Center Göttingen, Humboldtallee 23, 37073 Göttingen, Germany.
Abstract:
MicroRNAs (miRNAs) are important regulators of eukaryotic gene expression and their dysfunction is often associated with cancer. Alongside the canonical miRNA biogenesis pathway involving stepwise processing and export of pri- and pre-miRNA transcripts by the microprocessor complex, Exportin 5 and Dicer, several alternative mechanisms of miRNA production have been described. Here, we reveal that the atypical box C/D snoRNA U3, which functions as a scaffold during early ribosome assembly, is a miRNA source. We show that a unique stem-loop structure in the 5' domain of U3 is processed to form short RNA fragments that associate with Argonaute. miR-U3 production is independent of Drosha, and an increased amount of U3 in the cytoplasm in the absence of Dicer suggests that a portion of the full length snoRNA is exported to the cytoplasm where it is efficiently processed into miRNAs. Using reporter assays, we demonstrate that miR-U3 can act as a low proficiency miRNA in vivo and our data support the 3' UTR of the sortin nexin SNX27 mRNA as an endogenous U3-derived miRNA target. We further reveal that perturbation of U3 snoRNP assembly induces miR-U3 production, highlighting potential cross-regulation of target mRNA expression and ribosome production.
Insights
This study reveals that the U3 small nucleolar RNA (snoRNA) can produce microRNAs (miRNAs), termed miR-U3. This novel miRNA pathway bypasses canonical biogenesis and targets specific mRNAs, impacting gene expression and ribosome production.
Area of Science:
- Molecular Biology
- RNA Biology
- Gene Regulation
Background:
- MicroRNAs (miRNAs) are key regulators of gene expression, with dysregulation linked to cancer.
- Canonical miRNA biogenesis involves Drosha, Exportin 5, and Dicer.
- Alternative miRNA production pathways are increasingly recognized.
Purpose of the Study:
- To investigate the potential of small nucleolar RNAs (snoRNAs) as sources of miRNAs.
- To characterize the biogenesis and function of a novel miRNA derived from U3 snoRNA (miR-U3).
- To explore the implications of U3 snoRNA-derived miRNAs in gene regulation and cellular processes.
Main Methods:
- Analysis of RNA processing and localization.
- Argonaute association assays.
- Reporter assays to assess miRNA activity.
- Investigation of U3 snoRNP assembly perturbations.
Main Results:
- The atypical box C/D snoRNA U3 serves as a source for a novel miRNA, miR-U3.
- miR-U3 production is independent of Drosha and occurs via cytoplasmic processing of U3 snoRNA.
- miR-U3 exhibits low proficiency as a miRNA in vivo, targeting the SNX27 mRNA 3' UTR.
- Perturbation of U3 snoRNP assembly enhances miR-U3 production.
Conclusions:
- U3 snoRNA represents a previously unrecognized source of functional miRNAs.
- miR-U3 production offers a novel mechanism for post-transcriptional gene regulation.
- Cross-regulation between ribosome biogenesis and mRNA expression is mediated by U3 snoRNA-derived miRNAs.
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