Project Details
Projekt
Cellular imaging of RNA C-to-U editing
Applicants
Professor Dr. Jochen Meier; Professor Dr. Oliver Seitz
Subject Area
Biological and Biomimetic Chemistry
Term
from 2015 to 2019
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 277236491
RNA editing impacts RNA splicing and protein translation. The recent progress in high-throughput RNA sequencing has revealed an increasing number of mRNA editing sites. However, these methods provide an overview about the editing state of an ensemble of cells. Little is known about the dynamics of mRNA editing in single cells, the types of cells that undergo mRNA editing, the regulation of mRNA editing and the effect of editing on RNA localization. This lack of knowledge is probably caused by a paucity of suitable means for real-time RNA analysis in cells. Fluorescent imaging of RNA targets enable direct measurements - if desired - within living cells. However, the common RNA imaging techniques fail to provide the sequence specificity and sensitivity required for the analysis of RNA editing. We will close the methodological gap and develop fluorogenic oligonucleotide probes that afford fluorescent signals upon hybridization with matched (e.g. edited) mRNA but not upon hybridization with single nucleotide mismatched (e.g. unedited) mRNA. We will take advantage of Forced Intercalation (FIT) probe, in which a dye of the thiazole orange (TO) family of DNA intercalators replaces a canonical nucleobase. FIT-probes experience single nucleotide specific enhancements of fluorescent emission upon hybridization. To enable unambiguous nucleotide calls upon the RNA editing state two competing, differently colored FIT probes will be designed. One FIT probe will report the edited state (e.g. GlyRa2 c.656U), the other will inform about the unedited state (e.g. GlyRa2 c.656C). To improve sequence specificity and responsiveness we will introduce additional dyes and/or helper probes. Homo Fluorescence resonance energy transfer (FRET) will be designed to provide mismatch-responsive local energy sinks, wide-field excitation of hetero FRET systems will enable enhancements of brightness. The C-to-U RNA editing event studied in this research project confers a gain-of-function of the neurotransmitter receptor for glycine (GlyR) and, if up-regulated as in hippocampectomies of patients with epilepsy, may contribute to cognitive dysfunction and anxiety. This, however, depends on the neuron-type that expresses the RNA-edited GlyR. It is thus aim of the proposed research to obtain information about the dynamics and possible neuron type specificity of editing of the mRNA coding for GlyR. Initially we will investigate fixed primary neurons that ectopically express epitope tagged versions (HA and MS2 or BOTO) of edited and unedited GlyR. At an advanced stage, we will image neuronal endogenous GlyRF-coding RNA in fixed and living primary hippocampal neurons and acute slice preparations of our mouse model of epilepsy. Ultimately, this will enable single cell functional studies on the regulation of the RNA editing machinery.
DFG Programme
Priority Programmes
Cooperation Partner
Professor Dr. Martin Holtkamp
