RNA visualization in live bacterial cells using fluorescent protein complementation

Author: Valencia-Burton, M.; McCullough, R.M.; Cantor, C.R.; Broude, N.E.

Description: 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.

Subject headings: Aptamers, Nucleotide/chemistry; Escherichia coli/*metabolism; Eukaryotic Initiation Factor-4A/chemistry/genetics; Fluorescent Dyes/chemistry; Green Fluorescent Proteins/chemistry; Microscopy, Fluorescence; RNA/metabolism; RNA, Messenger/metabolism; RNA, Ribosomal, 5S/metabolism; RNA-Binding Proteins/genetics

Publication year: 2007

Journal or book title: Nature Methods

Volume: 4

Issue: 5

Pages: 421-427

Find the full text : https://www.nature.com/articles/nmeth1023

Find more like this one (cited by): https://scholar.google.com/scholar?cites=17435604579214649896&as_sdt=1000005&sciodt=0,16&hl=en

Type: Journal Article

Serial number: 2362