title: Riboswitch discovery by combining RNA-seq and genome-wide identification of transcriptional start sites.
abstract: Deep-sequencing technologies applied to RNA have tremendous potential to identify novel transcripts with single-nucleotide resolution. By combining whole-transcript cDNA sequencing (RNA-seq) and genome-wide identification of transcription start sites (dRNA-seq), it is possible to characterize long 5'-untranslated regions potentially endowed with regulatory capacities and to detect premature termination of transcription. This can be used to identify new potential riboswitches. In this chapter, we provide a detailed protocol of the dRNA-seq method based on differential pretreatment of RNAs with tobacco acid pyrophosphatase to differentiate between 5'-ends of primary and processed RNAs. We also give a briefer protocol of the preparation of RNA-seq libraries and of how to go through data bioinformatics analysis and data visualization using genome browsers. This approach is powerful to identify novel riboswitches and to demonstrate the functionality of riboswitches predicted in silico.
authors.affiliation: Unité de Biologie des Bactéries à Gram-Positif, Institut Pasteur, Paris, France; CNRS UMR 3525, Paris, France.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Rosinski-Chupin
authors.affiliation: Laboratoire Pathogenèse des Bactéries Anaérobies, Institut Pasteur, Paris, France; University of Paris Diderot, Sorbonne Paris Cité, Cellule Pasteur, Paris, France.
authors.forename: Olga
authors.initials: O
authors.lastname: Soutourina
authors.affiliation: Laboratoire Pathogenèse des Bactéries Anaérobies, Institut Pasteur, Paris, France; University of Paris Diderot, Sorbonne Paris Cité, Cellule Pasteur, Paris, France. Electronic address: isabelle.martin-verstraete@pasteur.fr.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Martin-Verstraete
mesh_terms: D020121:5' Untranslated Regions; D001419:Bacteria / Q000737:chemistry / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D058928:Riboswitch; D017423:Sequence Analysis, RNA / Q000379:methods*; D024363:Transcription Initiation Site
chemical_list: D020121:5' Untranslated Regions; D012333:RNA, Messenger; D058928:Riboswitch
keywords: 5′-untranslated region; Deep sequencing; RNA-seq; Riboswitches; Transcriptional start site mapping; cis-Acting regulatory RNA