Intracellular single molecule microscopy reveals two kinetically distinct pathways for microRNA assembly

被引：54

作者：

Pitchiaya, Sethuramasundaram ^{[1
]}

Androsavich, John R. ^{[1
,2
]}

Walter, Nils G. ^{[1
]}

机构：

[1] Univ Michigan, Dept Chem, Single Mol Anal Grp, Ann Arbor, MI 48109 USA

[2] Univ Michigan, Program Chem Biol, Ann Arbor, MI 48109 USA

来源：

EMBO REPORTS | 2012年 / 13卷 / 08期

基金：

美国国家卫生研究院;

关键词：

Let-7; miRNA; microinjection; single-molecule fluorescence microscopy; single-particle tracking; stepwise photobleaching; PROTEIN COMPLEXES; HUMAN-CELLS; IN-VIVO; DYNAMICS; LET-7;

D O I：

10.1038/embor.2012.85

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

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.

引用

页码：709 / 715

页数：7

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