title: Exploiting post-transcriptional regulation to probe RNA structures in vivo via fluorescence.
abstract: While RNA structures have been extensively characterized in vitro, very few techniques exist to probe RNA structures inside cells. Here, we have exploited mechanisms of post-transcriptional regulation to synthesize fluorescence-based probes that assay RNA structures in vivo. Our probing system involves the co-expression of two constructs: (i) a target RNA and (ii) a reporter containing a probe complementary to a region in the target RNA attached to an RBS-sequestering hairpin and fused to a sequence encoding the green fluorescent protein (GFP). When a region of the target RNA is accessible, the area can interact with its complementary probe, resulting in fluorescence. By using this system, we observed varied patterns of structural accessibility along the length of the Tetrahymena group I intron. We performed in vivo DMS footprinting which, along with previous footprinting studies, helped to explain our probing results. Additionally, this novel approach represents a valuable tool to differentiate between RNA variants and to detect structural changes caused by subtle mutations. Our results capture some differences from traditional footprinting assays that could suggest that probing in vivo via oligonucleotide hybridization facilitates the detection of folding intermediates. Importantly, our data indicate that intracellular oligonucleotide probing can be a powerful complement to existing RNA structural probing methods.
authors.affiliation: Microbiology Graduate Program, University of Texas at Austin, 100 E. 24th Street, A6500, Austin, TX 78712, USA.
authors.forename: Steven W
authors.initials: SW
authors.lastname: Sowa
authors.affiliation: McKetta Department of Chemical Engineering, University of Texas at Austin, 200 E. Dean Keeton St., Stop C0400, Austin, TX 78712, USA.
authors.forename: Jorge
authors.initials: J
authors.lastname: Vazquez-Anderson
authors.affiliation: McKetta Department of Chemical Engineering, University of Texas at Austin, 200 E. Dean Keeton St., Stop C0400, Austin, TX 78712, USA.
authors.forename: Chelsea A
authors.initials: CA
authors.lastname: Clark
authors.affiliation: McKetta Department of Chemical Engineering, University of Texas at Austin, 200 E. Dean Keeton St., Stop C0400, Austin, TX 78712, USA.
authors.forename: Ricardo
authors.initials: R
authors.lastname: De La Peña
authors.affiliation: McKetta Department of Chemical Engineering, University of Texas at Austin, 200 E. Dean Keeton St., Stop C0400, Austin, TX 78712, USA.
authors.forename: Kaitlin
authors.initials: K
authors.lastname: Dunn
authors.affiliation: McKetta Department of Chemical Engineering, University of Texas at Austin, 200 E. Dean Keeton St., Stop C0400, Austin, TX 78712, USA.
authors.forename: Emily K
authors.initials: EK
authors.lastname: Fung
authors.affiliation: McKetta Department of Chemical Engineering, University of Texas at Austin, 200 E. Dean Keeton St., Stop C0400, Austin, TX 78712, USA.
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Khoury
authors.affiliation: McKetta Department of Chemical Engineering, University of Texas at Austin, 200 E. Dean Keeton St., Stop C0400, Austin, TX 78712, USA lcontrer@che.utexas.edu.
authors.forename: Lydia M
authors.initials: LM
authors.lastname: Contreras
mesh_terms: D005456:Fluorescent Dyes; D005786:Gene Expression Regulation; D049452:Green Fluorescent Proteins / Q000235:genetics; D007438:Introns; D009154:Mutation; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization / Q000379:methods*; D015345:Oligonucleotide Probes; D012313:RNA / Q000737:chemistry*; D016337:RNA, Catalytic / Q000737:chemistry; D013768:Tetrahymena / Q000235:genetics
chemical_list: D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D016337:RNA, Catalytic; D049452:Green Fluorescent Proteins; D012313:RNA