title: In vivo expression and purification of aptamer-tagged small RNA regulators.
abstract: Small non-coding RNAs (sRNAs) are an emerging class of post-transcriptional regulators of bacterial gene expression. To study sRNAs and their potential protein interaction partners, it is desirable to purify sRNAs from cells in their native form. Here, we used RNA-based affinity chromatography to purify sRNAs following their expression as aptamer-tagged variants in vivo. To this end, we developed a family of plasmids to express sRNAs with any of three widely used aptamer sequences (MS2, boxB, eIF4A), and systematically tested how the aptamer tagging impacted on intracellular accumulation and target regulation of the Salmonella GcvB, InvR or RybB sRNAs. In addition, we successfully tagged the chromosomal rybB gene with MS2 to observe that RybB-MS2 is fully functional as an envelope stress-induced repressor of ompN mRNA following induction of sigmaE. We further demonstrate that the common sRNA-binding protein, Hfq, co-purifies with MS2-tagged sRNAs of Salmonella. The presented affinity purification strategy may facilitate the isolation of in vivo assembled sRNA-protein complexes in a wide range of bacteria.
authors.affiliation: RNA Biology Group, Max Planck Institute for Infection Biology, Charitéplatz 1, 10117 Berlin, Germany.
authors.forename: Nelly
authors.initials: N
authors.lastname: Said
authors.forename: Renate
authors.initials: R
authors.lastname: Rieder
authors.forename: Robert
authors.initials: R
authors.lastname: Hurwitz
authors.forename: Jochen
authors.initials: J
authors.lastname: Deckert
authors.forename: Henning
authors.initials: H
authors.lastname: Urlaub
authors.forename: Jörg
authors.initials: J
authors.lastname: Vogel
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry*; D001483:Base Sequence; D002846:Chromatography, Affinity; D002875:Chromosomes / Q000235:genetics; D008969:Molecular Sequence Data; D022661:RNA, Untranslated / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D052157:Aptamers, Nucleotide; D022661:RNA, Untranslated