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Published on: October 18, 2024
Development and Modification of Pre-miRNAs with a FRET Dye Pair for the Intracellular Visualization of Processing
Yukiko Kamiya1, Hiroshi Kamimoto1, Hongyu Zhu1
1Department of Biomolecular Engineering, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan.
Abstract:
microRNAs (miRNAs) are small non-coding ribonucleic acids (RNAs), which regulate gene expression via the RNA interference (RNAi) system. miRNAs have attracted enormous interest because of their biological significance and disease relationship. In cell systems, the generation of miRNA is regulated by multiple steps: the transfer of primary miRNA from the nucleus to the cytosol, the generation of the precursor-miRNA (pre-miRNA), the production of double-stranded RNA from pre-miRNA by the Dicer, the interaction with protein argonaute-2 (AGO2), and the subsequent release of one strand to form miRISC with AGO2. In this study, we attempt to visualize the intermediates that were generated in the miRNA-maturation step in the cells to acquire a detailed understanding of the maturation process of miRNA. To achieve this, we developed pre-miRNAs labeling with a Dicer- or AGO2-responsible fluorescence resonance energy transfer (FRET) dye pair. We observed that modifications with the dye at suitable positions did not interfere with the biological activities of pre-miRNAs. Further, imaging analyses employing these pre-miRNAs demonstrated that the processing of pre-miRNA promoted the accumulation of miRNA at the specific foci in the cytosol. The FRET-labeled pre-miRNA would further elucidate the mechanisms of the RNAi process and provide the basis for development of nucleic acid drugs working in the RNAi system.
Insights
Researchers visualized microRNA (miRNA) maturation intermediates using novel fluorescent dyes. This study reveals how precursor miRNA processing leads to miRNA accumulation in specific cellular locations, advancing RNA interference understanding.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- microRNAs (miRNAs) are key regulators of gene expression through the RNA interference (RNAi) pathway.
- Understanding miRNA biogenesis is crucial due to its role in biological processes and disease.
- Current knowledge of miRNA maturation intermediates in living cells remains limited.
Purpose of the Study:
- To visualize and understand the intermediates generated during miRNA maturation in real-time within cells.
- To investigate the dynamics of precursor miRNA processing and its impact on miRNA localization.
Main Methods:
- Development of pre-miRNAs labeled with a Dicer- or Argonaute-2 (AGO2)-specific fluorescence resonance energy transfer (FRET) dye pair.
- Assessment of dye modification impact on pre-miRNA biological activity.
- Cellular imaging analyses using FRET-labeled pre-miRNAs to observe miRNA maturation intermediates.
Main Results:
- Pre-miRNA labeling with FRET dyes did not impede their biological activity.
- Imaging revealed that precursor miRNA processing facilitates miRNA accumulation at specific cytosolic foci.
- The study provides a novel method for visualizing dynamic miRNA maturation events.
Conclusions:
- FRET-labeled pre-miRNAs offer a powerful tool to study the intricate mechanisms of miRNA biogenesis.
- The findings enhance our understanding of the RNAi pathway and miRNA processing dynamics.
- This approach lays the groundwork for developing novel nucleic acid-based therapeutics targeting the RNAi system.
