A FRET-based assay for characterization of alternative splicing events using peptide nucleic acid fluorescence in situ hybridization.
We describe a quantitative method for detecting RNA alternative splicing variants that combines in situ hybridization of fluorescently labeled peptide nucleic acid (PNA) probes with confocal microscopy Förster resonance energy transfer (FRET). The use of PNA probes complementary to sequences flanking a given splice junction allows to specifically quantify, within the cell, the RNA isoform generating such splice junction by FRET measure. As a proof of concept we analyzed two alternative splicing events originating from lymphocyte antigen 6 (LY6) complex, locus G5B (LY6G5B) pre-mRNA. These are characterized by the removal of the first intron (Fully Spliced Isoform, FSI) or by retention of such intron (Intron-Retained Isoform, IRI). The use of PNA probe pairs labeled with donor (Cy3) and acceptor (Cy5) fluorophores, suitable to FRET, flanking FSI and IRI specific splice junctions specifically detected both mRNA isoforms in HeLa cells. We have observed that the method works efficiently with probes 5-11 nt apart. The data supports that this FRET-based PNA fluorescence in situ hybridization (FP-FISH) method offers a conceptually new approach for characterizing at the subcellular level not only splice variant isoform structure, location and dynamics but also potentially a wide variety of close range RNA-RNA interactions.
Blanco
Ana M
AM
Sistemas Genómicos S.L., Parque Tecnológico de Valencia, Ronda G. Marconi 6, E-46980 Paterna, Spain.
Rausell
Laura
L
Aguado
Begoña
B
Perez-Alonso
Manuel
M
Artero
Rubén
R
D017398:Alternative Splicing; D002466:Cell Nucleolus / Q000737:chemistry; D003593:Cytoplasm / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer; D006367:HeLa Cells; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D018613:Microscopy, Confocal; D015341:Nucleic Acid Probes / Q000737:chemistry*; D010434:Pepsin A; D020135:Peptide Nucleic Acids / Q000737:chemistry*; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis
D015341:Nucleic Acid Probes; D020135:Peptide Nucleic Acids; D020033:Protein Isoforms; D012333:RNA, Messenger; D010434:Pepsin A