title: Experimental discovery of sRNAs in Vibrio cholerae by direct cloning, 5S/tRNA depletion and parallel sequencing.
abstract: Direct cloning and parallel sequencing, an extremely powerful method for microRNA (miRNA) discovery, has not yet been applied to bacterial transcriptomes. Here we present sRNA-Seq, an unbiased method that allows for interrogation of the entire small, non-coding RNA (sRNA) repertoire in any prokaryotic or eukaryotic organism. This method includes a novel treatment that depletes total RNA fractions of highly abundant tRNAs and small subunit rRNA, thereby enriching the starting pool for sRNA transcripts with novel functionality. As a proof-of-principle, we applied sRNA-Seq to the human pathogen Vibrio cholerae. Our results provide information, at unprecedented depth, on the complexity of the sRNA component of a bacterial transcriptome. From 407 039 sequence reads, all 20 known V. cholerae sRNAs, 500 new, putative intergenic sRNAs and 127 putative antisense sRNAs were identified in a limited number of growth conditions examined. In addition, characterization of a subset of the newly identified transcripts led to the identification of a novel sRNA regulator of carbon metabolism. Collectively, these results strongly suggest that the number of sRNAs in bacteria has been greatly underestimated and that future efforts to analyze bacterial transcriptomes will benefit from direct cloning and parallel sequencing experiments aided by 5S/tRNA depletion.
authors.affiliation: HHMI, Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, MA 02111, USA.
authors.forename: Jane M
authors.initials: JM
authors.lastname: Liu
authors.forename: Jonathan
authors.initials: J
authors.lastname: Livny
authors.forename: Michael S
authors.initials: MS
authors.lastname: Lawrence
authors.forename: Marc D
authors.initials: MD
authors.lastname: Kimball
authors.forename: Matthew K
authors.initials: MK
authors.lastname: Waldor
authors.forename: Andrew
authors.initials: A
authors.lastname: Camilli
mesh_terms: D001483:Base Sequence; D002244:Carbon / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods*; D008969:Molecular Sequence Data; D012329:RNA, Bacterial / Q000032:analysis / Q000737:chemistry* / Q000235:genetics; D012341:RNA, Ribosomal, 5S / Q000737:chemistry; D012343:RNA, Transfer / Q000737:chemistry; D022661:RNA, Untranslated / Q000032:analysis / Q000737:chemistry* / Q000235:genetics; D017423:Sequence Analysis, RNA / Q000379:methods*; D012689:Sequence Homology, Nucleic Acid; D014734:Vibrio cholerae / Q000235:genetics* / Q000378:metabolism
chemical_list: D012329:RNA, Bacterial; D012341:RNA, Ribosomal, 5S; D022661:RNA, Untranslated; D002244:Carbon; D012343:RNA, Transfer