RRC ID 53857
Author Ando H, Hirose M, Kurosawa G, Impey S, Mikoshiba K.
Title Time-lapse imaging of microRNA activity reveals the kinetics of microRNA activation in single living cells.
Journal Sci Rep
Abstract MicroRNAs (miRNAs) are small, non-coding RNAs that play critical roles in the post-transcriptional regulation of gene expression. Although the molecular mechanisms of the biogenesis and activation of miRNA have been extensively studied, the details of their kinetics within individual living cells remain largely unknown. We developed a novel method for time-lapse imaging of the rapid dynamics of miRNA activity in living cells using destabilized fluorescent proteins (dsFPs). Real-time monitoring of dsFP-based miRNA sensors revealed the duration necessary for miRNA biogenesis to occur, from primary miRNA transcription to mature miRNA activation, at single-cell resolution. Mathematical modeling, which included the decay kinetics of the fluorescence of the miRNA sensors, demonstrated that miRNAs induce translational repression depending on their complementarity with targets. We also developed a dual-color imaging system, and demonstrated that miR-9-5p and miR-9-3p were produced and activated from a common hairpin precursor with similar kinetics, in single cells. Furthermore, a dsFP-based miR-132 sensor revealed the rapid kinetics of miR-132 activation in cortical neurons under physiological conditions. The timescale of miRNA biogenesis and activation is much shorter than the median half-lives of the proteome, suggesting that the degradation rates of miRNA target proteins are the dominant rate-limiting factors for miRNA-mediated gene silencing.
Volume 7(1)
Pages 12642
Published 2017-10-3
DOI 10.1038/s41598-017-12879-2
PII 10.1038/s41598-017-12879-2
PMID 28974737
PMC PMC5626736
MeSH Gene Expression Regulation Humans Kinetics MicroRNAs / biosynthesis MicroRNAs / genetics* RNA Stability / genetics Single-Cell Analysis / methods* Time-Lapse Imaging / methods*
IF 3.998
Times Cited 5
DNA material pdsGFP (RDB15992) pdsCFP (RDB15993) pdsVenus (RDB15994)