RNA visualization in live bacterial cells using fluorescent protein complementation.
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.
Valencia-Burton
Maria
M
Center for Advanced Biotechnology and Department of Biomedical Engineering, Boston University, 36 Cummington St., Boston, Massachusetts 02215, USA.
McCullough
Ron M
RM
Cantor
Charles R
CR
Broude
Natalia E
NE
D052157:Aptamers, Nucleotide / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism*; D039601:Eukaryotic Initiation Factor-4A / Q000737:chemistry / Q000235:genetics*; D005456:Fluorescent Dyes / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000737:chemistry*; D008856:Microscopy, Fluorescence; D012313:RNA / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D012341:RNA, Ribosomal, 5S / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics*
D052157:Aptamers, Nucleotide; D005456:Fluorescent Dyes; D012333:RNA, Messenger; D012341:RNA, Ribosomal, 5S; D016601:RNA-Binding Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D012313:RNA; D039601:Eukaryotic Initiation Factor-4A