Probing microRNA Activity in vitro and inside Cells using Single Molecule Microscopy.

Abstract

Non-coding RNAs (ncRNAs) outnumber their protein-coding counterparts, yet their presumably diverse functions are still ill-understood. This thesis reports the development of two novel single-molecule methods to probe the activity of microRNAs (miRNAs), a class of regulatory ncRNAs, in pursuit of their elusive mechanism of action.

miRNAs associate with components of the RNA induced silencing complex (RISC) to assemble on messenger RNA (mRNA) targets and regulate protein expression in higher eukaryotes. Here, I describe a method for the intracellular single molecule, high resolution localization and counting (iSHiRLoC) of miRNAs. Microinjected, singly fluorophore labeled, functional miRNAs were tracked within diffusing particles, a majority of which contained single miRNA molecules. Observed mobility and mRNA dependent assembly changes support a model of multiple target turnovers by miRNAs, revealing the dynamic nature of an important gene regulatory pathway and paving the way towards its single molecule systems biology.

miRNAs accumulate in processing bodies (PBs), sub-cellular foci enriched in RNA processing enzymes, as a cause or consequence of post-transcriptional gene silencing. Despite numerous observations, quantitative analysis of miRNA localization within PBs has been lacking. iSHiRLoC of miRNAs revealed that only a small fraction of miRNAs localized to PBs and majority of PBs contained only one or two fluorophore labeled miRNA molecules. Moreover, miRNAs resided in PBs only for a few hundred milliseconds, suggesting the preponderance of unstable interactions. Heterogeneous distribution of miRNAs in PBs coupled with the observation that miRNAs docked onto PBs either stably or transiently suggests an underlying diversity in the composition of these two complexes.

Some mRNAs contain multiple binding sites for a specific miRNA, presumably for enhanced regulation. To quantify the binding stoichiometry between miRNAs and such mRNAs, I developed a single-molecule in vitro assay based on the step-wise photobleaching of fluorescent probes. Our data, in two different cell extracts, showed that a majority of mRNAs are either bound by zero or a single miRNA under conditions of maximal repression. Computational mRNA structure analysis predicted low accessibility of miRNA binding sites. Together, these data suggest that a higher number of binding sites corresponds to a higher probability of binding, not multiple occupancy.