Direct detection of RNA transcription by FRET imaging using fluorescent protein probe.
We have constructed a reporter system for intracellular direct detection of RNA transcription that consists of two biomolecular components. The first part is a GFP-based recombinant protein probe (YRG0C-11ad) containing the RNA-binding Rev-peptide between ECFP and EYFP. The second component is RRE-RNA, which specifically binds to the Rev-peptide. Cells stably expressing YRG0C-11ad were identified by an increased FRET signal after direct transfection or intracellular transcription of RRE-RNA. In addition, the signal increase is more noticeable if tandemly repeated RRE-RNA is used as the reporter. Untranslatable non-coding RNAs are regarded as regulators of cellular gene expression, but they are difficult to study using indirect reporter systems that are dependent on translational products. Direct detection of reporter RNA would be a useful method for the detection of intracellular promoter activity during transcription of untranslatable RNAs.
Endoh
Tamaki
T
Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama 226-8501, Japan.
Mie
Masayasu
M
Kobatake
Eiry
E
D001483:Base Sequence; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008954:Models, Biological; D011485:Protein Binding; D012313:RNA / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D012313:RNA