Talk abstracts

Talk on Saturday 11:45-12:00pm submitted by Sethu Pitchiaya

Small RNAs, Big Impact: Probing microRNA Activity inside Human Cells Using Single Molecule Microscopy

Sethuramasundaram Pitchiaya (Department of Chemistry, University of Michigan, Ann Arbor, MI 48109-1055), John R. Androsavich (Program in Chemical Biology, University of Michigan, Ann Arbor, MI 48109-1055), Nils G. Walter (Department of Chemistry, University of Michigan, Ann Arbor, MI 48109-1055)

Abstract:
microRNA (miRNA) mediated gene regulation is an evolutionarily conserved cellular RNA interference (RNAi) pathway wherein cellular non-(protein)coding RNAs ~22-nucleotide in length, associated with a cohort of proteins, bind to partially complementary regions on messenger RNA (mRNA) targets to repress protein expression. While standard ensemble assays, including intracellular fluorescence microscopy, have revealed a wealth of information on miRNA function and intracellular localization, the mechanism of miRNA mediated mRNA repression is still under intense debate. We have developed a novel method to investigate miRNA activity inside living (and fixed) human cells using single particle tracking and single molecule fluorescence microscopy with 30 nm spatial accuracy and 100 ms time resolution. Upon microinjection into the cytoplasm of HeLa cells, fluorophore labeled repression competent mature miRNAs were visualized as distinct diffraction limited particles freely diffusing with multifarious diffusive behaviors. More specifically, two major populations particles bearing diffusion coefficients (0.26 µm2/s and 0.034 µm2/s) closely resembling those of messenger RNPs and processing bodies were obtained. The number of miRNA molecules per fluorescent particle was then quantified using the stochastic photobleaching property of fluorescent probes in formaldehyde fixed cells. A majority of miRNA particles contained single miRNA molecules, however, a significant proportion of spots had more than one molecule (up to seven). Time dependent changes in miRNA diffusion and distribution were also observed in live and fixed cells respectively, suggesting a miRNA multiple turnover model. These results demonstrate the ability of our method to unravel the dynamic nature of an important gene regulatory pathway, and the potential for it to be extended to many other small, non-coding RNAs.

Keywords: microRNA, Single particle tracking microscopy, Single molecule fluorescence microscopy