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Intracellular single molecule microscopy reveals two kinetically distinct pathways for microRNA assembly
Author(s) -
Pitchiaya Sethuramasundaram,
Androsavich John R,
Walter Nils G
Publication year - 2012
Publication title -
embo reports
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 4.584
H-Index - 184
eISSN - 1469-3178
pISSN - 1469-221X
DOI - 10.1038/embor.2012.85
Subject(s) - intracellular , microbiology and biotechnology , microrna , biophysics , chemistry , biology , nanotechnology , biochemistry , gene , materials science
MicroRNAs (miRNAs) associate with components of the RNA‐induced silencing complex (RISC) to assemble on mRNA targets and regulate protein expression in higher eukaryotes. Here we describe a method for the intracellular single‐molecule, high‐resolution localization and counting (iSHiRLoC) of miRNAs. Microinjected, singly fluorophore‐labelled, functional miRNAs were tracked within diffusing particles, a majority of which contained single such miRNA molecules. Mobility and mRNA‐dependent assembly changes suggest the existence of two kinetically distinct pathways for miRNA assembly, revealing the dynamic nature of this important gene regulatory pathway. iSHiRLOC achieves an unprecedented resolution in the visualization of functional miRNAs, paving the way to understanding RNA silencing through single‐molecule systems biology.