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

Abstract

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.

KRAS Engages AGO2 to Enhance Cellular Transformation

“…Diffusion coefficients of microinjected fluorophore labeled let-7a molecules suggest that, in NIH3T3 cells expressing KRAS WT , let-7a assembles into both ‘fast’ (low molecular weight) and ‘slow’ (high molecular weight) mRNA-protein complexes (mRNPs; Figure 6G and S6M). By contrast, in cells expressing KRAS G12V , let-7a manifested predominantly in fast moving complexes, suggesting that let-7a is unable to accumulate in larger mRNPs (known to be functional RISC; (Pitchiaya et al, 2012; Pitchiaya et al, 2013)) in an oncogenic KRAS setting. Importantly, in cells expressing KRAS G12VY64G , let-7a accumulates in both fast and slow mRNPs, further implicating that a direct interaction between mutant KRAS and AGO2 is essential to prevent functional RISC assembly.…”

“…To more directly explore the potential effect of KRAS G12V on functional messenger ribonucleoprotein particles (mRNPs), we exploited a recently described method for intracellular single-molecule, high-resolution localization and counting (iSHiRLoC) of microRNAs (Pitchiaya et al, 2012; Pitchiaya et al, 2013). Diffusion coefficients of microinjected fluorophore labeled let-7a molecules suggest that, in NIH3T3 cells expressing KRAS WT , let-7a assembles into both ‘fast’ (low molecular weight) and ‘slow’ (high molecular weight) mRNA-protein complexes (mRNPs; Figure 6G and S6M).…”

A novel method to accurately locate and count large numbers of steps by photobleaching

“…By contrast, photobleaching event counting holds the promise to quantify release of DDS cargo more accurately (Pitchiaya et al. , 2012, 2013, 2014; Shankar et al. , 2016).…”

Single-molecule tools for enzymology, structural biology, systems biology and nanotechnology: an update

“…We recently reported on the temporal assembly of miRNP complexes using a technique we termed intracellular single-molecule, high resolution localization and counting, or iSHiRLoC (Fig. 1d) (Pitchiaya et al 2012; Pitchiaya et al 2013). By combining live and fixed cell imaging of microinjected, cyanine dye-labeled miRNAs in HeLa cells, we were able to track miRNP assembly and count the number of miRNAs within single particles (Fig.…”

“…c Example experimental setup and fluorescence intensity trace for an experiment investigating binding and unbinding of fluorescently-labeled probes to an immobilized oligonucleotide. d Example experimental setup and data for an intracellular single-molecule high resolution localization and counting (iSHiRLoC) experiment investigating microRNA diffusion in live cells (Johnson-Buck and Walter 2014; Pitchiaya et al 2013)…”