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Updated: Oct 21, 2025
Genome-wide Screen for miRNA Targets Using the MISSION Target ID Library
Published on: April 6, 2012
m6A-seq analysis of microRNAs reveals that the N6-methyladenosine modification of miR-21-5p affects its target
Hanming Wang1, Xinyun Song1, Chun Song1
1Laboratory of Nucleic Acid Technology, Institute of Molecular Medicine, Peking University, Beijing, 100871, China.
Abstract:
In eukaryotes, N6-methyladenosine (m6A) is one of the most abundant modifications on RNAs, and it plays important roles in many biological processes and diseases such as cancer. While most m6A researches focus on message RNAs and long non-coding RNAs, recent studies have reported the presence of m6A in small RNAs. Nevertheless, current knowledge about m6A prevalence in mature microRNAs (miRNA) is extremely limited and the functional significance of m6A methylation in miRNAs remains to be elucidated. Here, we demonstrated cell-specific m6A profiles of miRNAs in A549 human non-small cell lung cancer (NSCLC) cells and HEK293A cells by using miRNA m6A immunoprecipitation sequencing and constructed the consensus motif in m6A-enriched miRNAs de novo. We found that miR-21-5p, an oncogenic miRNA, showed the highest m6A enrichment in NSCLC cells. Depletion of the demethylase ALKBH5 did not change the expression level of miR-21-5p, but altered the m6A abundance of miR-21-5p, thereby changing the expression levels of its target gene. We further synthesized m6A modified miR-21-5p mimics in vitro and demonstrated that in NSCLC cells, m6A marks in mature miR-21-5p could directly affect its silencing potency towards target genes, which finally impaired its promotion to proliferation and motility. Together, our findings reveal the landscape of m6A modification in mature miRNAs, and provide the first evidence that it may contribute to the mRNA responses to cancer-related miRNAs.
Insights
N6-methyladenosine (m6A) modification in mature microRNAs (miRNAs) impacts cancer cell behavior. This study reveals m6A on miR-21-5p affects its gene silencing, influencing proliferation and motility in non-small cell lung cancer.
Area of Science:
- Epigenetics
- RNA Biology
- Cancer Research
Background:
- N6-methyladenosine (m6A) is a prevalent RNA modification in eukaryotes, crucial for biological processes and diseases like cancer.
- While m6A in mRNA and lncRNA is studied, its presence and function in mature microRNAs (miRNAs) are largely unknown.
- Understanding m6A in miRNAs is vital for elucidating their roles in cellular functions and disease pathogenesis.
Purpose of the Study:
- To investigate the prevalence and functional significance of m6A modification in mature miRNAs.
- To characterize cell-specific m6A profiles in human non-small cell lung cancer (NSCLC) cells.
- To determine the impact of m6A on the oncogenic miRNA miR-21-5p and its target genes.
Main Methods:
- miRNA m6A immunoprecipitation sequencing (miRIP-seq) to profile m6A enrichment in miRNAs.
- De novo construction of consensus motifs for m6A-modified miRNAs.
- Depletion of the demethylase ALKBH5 to assess its effect on miRNA m6A levels and target gene expression.
- In vitro synthesis of m6A-modified miR-21-5p mimics for functional assays.
Main Results:
- Cell-specific m6A profiles of mature miRNAs were established in A549 NSCLC and HEK293A cells.
- The oncogenic miR-21-5p exhibited the highest m6A enrichment in NSCLC cells.
- ALKBH5 depletion altered miR-21-5p m6A abundance, impacting target gene expression.
- m6A modification in mature miR-21-5p directly influenced its silencing potency, affecting cancer cell proliferation and motility.
Conclusions:
- This study reveals the landscape of m6A modification in mature miRNAs.
- Provides the first evidence that m6A methylation in mature miRNAs, specifically miR-21-5p, directly impacts gene silencing and cancer-related phenotypes.
- Highlights a novel regulatory mechanism of miRNA function in cancer progression.
