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Pitchiaya S.,Smart USA | Pitchiaya S.,Single Molecule Analysis Group | Heinicke L.A.,Single Molecule Analysis Group | Custer T.C.,University of Michigan | And 2 more authors.
Chemical Reviews | Year: 2014

Numerous RNAs that are found in particularly the eukaryotic cell and a detailed summary of the single molecule tools available to study RNAs intracellularly is reviewed. The majority of RNAs found in the eukaryotic cell do not code for protein but rather regulate gene expression via post-transcriptional gene silencing or epigenetic gene regulation. Intracellular single molecule techniques offer an unparalleled means to investigate the behavior of these emerging classes of non-coding RNAs (ncRNAs). Many RNA elements have been found to originate from overlapping loci, suggesting that similar RNA sequences can be distinctly generated or processed to perform different biological functions. RNA-seq technologies coupled with RNA-protein cross-linking have been successful in mapping RNA target binding sites of RNA-binding proteins on a genomic scale. miRNAs are the most ubiquitous small ncRNA in humans, with over 1500 different mammalian miRNA sequences discovered that represent more than 1% of the entire genome and thus the largest gene family.

Pitchiaya S.,Single Molecule Analysis Group | Androsavich J.R.,Single Molecule Analysis Group | Androsavich J.R.,University of Michigan | Walter N.G.,Single Molecule Analysis Group
EMBO Reports | Year: 2012

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. © 2012 European Molecular Biology Organization.

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