Dissecting non-coding RNA mechanisms in cellulo by Single-molecule High-Resolution Localization and Counting

作者：Pitchiaya Sethuramasundaram; Krishnan Vishalakshi; Custer Thomas C; Walter Nils G^*

来源：Methods, 2013, 63(2): 188-199.

DOI：10.1016/j.ymeth.2013.05.028

• RNA silencing
• microRNA
• Non-coding RNA
• Single molecule microscopy
• Single particle tracking

Non-coding RNAs (ncRNAs) recently were discovered to outnumber their protein-coding counterparts, yet their diverse functions are still poorly understood. Here we report on a method for the intracellular Single-molecule High-Resolution Localization and Counting (iSHiRLoC) of microRNAs (miRNAs), a conserved, ubiquitous class of regulatory ncRNAs that controls the expression of over 60% of all mammalian protein coding genes post-transcriptionally, by a mechanism shrouded by seemingly contradictory observations. We present protocols to execute single particle tracking (SPT) and single-molecule counting of functional microinjected, fluorophore-labeled miRNAs and thereby extract diffusion coefficients and molecular stoichiometries of micro-ribonucleoprotein (miRNP) complexes from living and fixed cells, respectively. This probing of miRNAs at the single molecule level sheds new light on the intracellular assembly/disassembly of miRNPs, thus beginning to unravel the dynamic nature of this important gene regulatory pathway and facilitating the development of a parsimonious model for their obscured mechanism of action.

• 出版日期2013-9-15