Abstract
RNA localization in subcellular compartments is essential for spatial and temporal regulation of protein expression in neurons. Several techniques have been developed to visualize mRNAs inside cells, but the study of the behavior of endogenous and nonengineered mRNAs in living neurons has just started. In this study, we combined reduction-triggered fluorescent (RETF) probes and fluorescence correlation spectroscopy (FCS) to investigate the diffusion properties of activity-regulated cytoskeleton-associated protein (Arc) and inositol 1,4,5-trisphosphate receptor type 1 (Ip3r1) mRNAs. This approach enabled us to discriminate between RNA-bound and unbound fluorescent probes and to quantify mRNA diffusion parameters and concentrations in living rat primary hippocampal neurons. Specifically, we detected the induction of Arc mRNA production after neuronal activation in real time. Results from computer simulations with mRNA diffusion coefficients obtained in these analyses supported the idea that free diffusion is incapable of transporting mRNA of sizes close to those of Arc or Ip3r1 to distal dendrites. In conclusion, the combined RETF-FCS approach reported here enables analyses of the dynamics of endogenous, unmodified mRNAs in living neurons, affording a glimpse into the intracellular dynamics of RNA in live cells.
Highlights
RNA localization in subcellular compartments is essential for spatial and temporal regulation of protein expression in neurons
Fluorescence derived from reduction-triggered fluorescent (RETF) probes increases after introduction by patch clamp We designed two RETF probes targeting different mRNA locations, termed target 1 and target 2, for activity-regulated cytoskeleton protein (Arc) mRNA and Ip3r1 mRNA, respectively
To test whether the RETF probes can detect target RNAs in living neurons, we introduced sets of RETF probes targeting Arc mRNA or Ip3r1 mRNA into hippocampal neurons by the whole-cell patch-clamp method
Summary
RNA localization in subcellular compartments is essential for spatial and temporal regulation of protein expression in neurons. The MS2 system is the most popular one and enables highly sensitive and specific imaging of transcripts [6] This method requires genetic manipulation of the target RNA sequence by inserting multiple aptamer-binding sequences. Switch-on fluorogenic hybridization probes have been used to investigate the dynamics of unmodified mRNAs. The peptidic Pumilio homology domain RNA probe binds to RNA in a sequence-specific manner by recognizing eight conterminous bases, allowing spatiotemporal analysis of endogenous RNA localization [9, 10]. The peptidic Pumilio homology domain RNA probe binds to RNA in a sequence-specific manner by recognizing eight conterminous bases, allowing spatiotemporal analysis of endogenous RNA localization [9, 10] It requires optimization of the amino acid sequence for each target. Quantification of mRNA dynamics by FCS and RETF probe fluorescence derived from reduced probes dissociated from target mRNA complicates quantitative analyses
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