Talk on Friday 04:42-04:54pm submitted by Laurie Heinicke
Dissecting miRNA function and localization using single molecule approaches
Laurie A. Heinicke (Chemistry, University of Michigan), Sethuramasundaram Pitchiaya (Chemistry, University of Michigan), Elizabeth L. Cameron (Chemistry, University of Michigan), Nils G. Walter (Chemistry, University of Michigan)
MicroRNA (miRNA) genes are transcribed in the nucleus as primary miRNA transcripts (pri-miRNA) and processed via multiple steps to generate mature miRNAs in the cytoplasm. These small non-coding RNA (ncRNAs) associate with components of the RNA-induced silencing complex (RISC) and engage mRNA targets and regulate gene repression via translational inhibition and/or mRNA degradation. Despite rapid advances in our understanding of miRNA biogenesis and mechanism, the intracellular dynamics and assembly of miRNA-associated complexes and the spatiotemporal modulation of miRNA-regulated gene expression remain unclear. To uniquely probe intracellular RNA silencing pathways, our lab has developed a method termed intracellular Single-molecule High-Resolution Localization and Counting (iSHiRLoC) to determine the localization, diffusion constant and assembly state of single miRNA complexes inside living human cells at 30 nm spatial and 100 ms temporal resolution. We have extended the capabilities of iSHiRLoC to simultaneously detect two colors and examined the diffusion and co-localization of microinjected fluorophore-labeled miRNAs and fluorescent protein labeled P-bodies, sub-cellular foci enriched in RNA degrading enzymes. Our data indicate that there are at least two types of temporarily increasing miRNAs that reside in P-bodies for ~1.18 s and >10 s, representing transient and tight association with P-bodies, respectively, wherein the latter likely reflects miRNA-directed mRNA degradation. In addition, we have used iSHiRLoC to probe nuclear functions of miRNAs. We find a significant fraction of microinjected mature let-7-a1 localizes to the nucleus, whereas artificial cxcr4 miRNA localizes to the nucleus at a much lower extent. Inhibition of transcription significantly reduces let-7-a1 nuclear localization, similar to the level of cxcr4 in the absence or presence of transcription inhibitor, suggesting that only let-7-a1 binds RNA targets in the nucleus. We are currently pursuing complementary biochemical approaches, such as subcellular fractionation and Northern blotting, to further validate our iSHiRLoC observations. Together, these studies highlight the versatility of iSHiRLoC by providing biological insight regarding assembly and localization of intracellular miRNAs.
Keywords: miRNA, single molecule , fluorescence microscopy