RNA visualization in live bacterial cells using fluorescent protein complementation

Abstract

We describe a technique for the detection and localization of RNA transcripts in living cells. The method is based on fluorescent-protein complementation regulated by the interaction of a split RNA-binding protein with its corresponding RNA aptamer. In our design, the RNA-binding protein is the eukaryotic initiation factor 4A (eIF4A). eIF4A is dissected into two fragments, and each fragment is fused to split fragments of the enhanced green fluorescent protein (EGFP). Coexpression of the two protein fusions in the presence of a transcript containing eIF4A-interacting RNA aptamer resulted in the restoration of EGFP fluorescence in Escherichia coli cells. We also applied this technique to the visualization of an aptamer-tagged mRNA and 5S ribosomal RNA (rRNA). We observed distinct spatial and temporal changes in fluorescence within single cells, reflecting the nature of the transcript.

A FACS-based screening strategy to assess sequence-specific RNA-binding of Pumilio protein variants in E. coli

“…Based on our in vitro data, we know that TetFC is not prone to self-complementation, reducing the number of false positives. It has to be stated, however, that our in vivo TetFC shows a rather low signal increase compared to related work on RNA-dependent fluorescence complementation systems in E. coli analyzed via flow cytometry (Valencia-Burton et al, 2007Yiu et al, 2011). This is most likely due to the number of components required for GFP complementation.…”

Aptamer-based molecular imaging

“…mRNA transport was also coupled to translation repression during transport (Rackham and Brown, 2004) and regulated local translation (Bi et al, 2006;Daigle and Ellenberg, 2007;Dictenberg et al, 2008). Finally, the aptamer-fluorescent protein imaging systems (MS2-GFP and split eIF4A-EGFP) was applied to show the differential spatiotemporal localization of E. coli RNAs (Valencia-Burton et al, 2007;Valencia-Burton et al, 2009;Nevo-Dinur et al, 2011), the pri-miRNA localization in D-body in Arabidopsis (Fang and Spector, 2007) and the binding dynamics of noncoding Xist RNA to X-chromosome (Ng et al, 2011).…”

Recent progress in the construction methodology of fluorescent biosensors based on biomolecules

“…(Ghosh, I. et al 2000) Based on this strategy, a receptor composed of two subunits that are associated by binding to the analyte can be converted into a fluorescent biosensor by connecting each of the two subunits with each split AFP fragment (Figure 2). Actually, several types of biosensors have been developed for fluorescent detection of specific DNA sequences (Stains, C. I. et al 2005;Demidov, V. V. et al 2006), DNA methylation (Stains, C. I. et al 2006), mRNA (Ozawa, T. et al 2007;Valencia-Burton, M. et al 2007) and protein interactions (Nyfeler, B. et al 2005;Hu, C. -D. et al 2003;Wilson, C. G. et al, 2004). Unlike the above-mentioned split AFP reconstitution, in which split AFP halves reform into a fluorescent structure via noncovalent association, another reconstitution strategy, inteinmediated reconstitution, has been developed by Ozawa and co-workers .…”