title: RNA Sequencing Identifies New RNase III Cleavage Sites inEscherichia coliand Reveals Increased Regulation of mRNA.
abstract: Ribonucleases facilitate rapid turnover of RNA, providing cells with another mechanism to adjust transcript and protein levels in response to environmental conditions. While many examples have been documented, a comprehensive list of RNase targets is not available. To address this knowledge gap, we compared levels of RNA sequencing coverage ofEscherichia coliand a corresponding RNase III mutant to expand the list of known RNase III targets. RNase III is a widespread endoribonuclease that binds and cleaves double-stranded RNA in many critical transcripts. RNase III cleavage at novel sites found inaceEF,proP,tnaC,dctA,pheM,sdhC,yhhQ,glpT,aceK, andgluQaccelerated RNA decay, consistent with previously described targets wherein RNase III cleavage initiates rapid degradation of secondary messages by other RNases. In contrast, cleavage at three novel sites in theahpF,pflB, andyajQtranscripts led to stabilized secondary transcripts. Two other novel sites inhisLandpheMoverlapped with transcriptional attenuators that likely serve to ensure turnover of these highly structured RNAs. Many of the new RNase III target sites are located on transcripts encoding metabolic enzymes. For instance, two novel RNase III sites are located within transcripts encoding enzymes near a key metabolic node connecting glycolysis and the tricarboxylic acid (TCA) cycle. Pyruvate dehydrogenase activity was increased in anrncdeletion mutant compared to the wild-type (WT) strain in early stationary phase, confirming the novel link between RNA turnover and regulation of pathway activity. Identification of these novel sites suggests that mRNA turnover may be an underappreciated mode of regulating metabolism.IMPORTANCEThe concerted action and overlapping functions of endoribonucleases, exoribonucleases, and RNA processing enzymes complicate the study of global RNA turnover and recycling of specific transcripts. More information about RNase specificity and activity is needed to make predictions of transcript half-life and to design synthetic transcripts with optimal stability. RNase III does not have a conserved target sequence but instead recognizes RNA secondary structure. Prior to this study, only a few RNase III target sites inE. coliwere known, so we used RNA sequencing to provide a more comprehensive list of cleavage sites and to examine the impact of RNase III on transcript degradation. With this added information on how RNase III participates in transcript regulation and recycling, a more complete picture of RNA turnover can be developed forE. coliSimilar approaches could be used to augment our understanding of RNA turnover in other bacteria.
authors.affiliation: Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, Wisconsin, USA.|Microbiology Doctoral Training Program, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Gina C
authors.initials: GC
authors.lastname: Gordon
authors.affiliation: Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Jeffrey C
authors.initials: JC
authors.lastname: Cameron
authors.affiliation: Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, Wisconsin, USA pfleger@engr.wisc.edu.|Microbiology Doctoral Training Program, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Brian F
authors.initials: BF
authors.lastname: Pfleger
mesh_terms: D004926:Escherichia coli / Q000201:enzymology*; D015964:Gene Expression Regulation, Bacterial; D006868:Hydrolysis; D020871:RNA Stability; D012333:RNA, Messenger / Q000378:metabolism*; D043244:Ribonuclease III / Q000737:chemistry / Q000378:metabolism*; D017423:Sequence Analysis, RNA; D013379:Substrate Specificity
chemical_list: D012333:RNA, Messenger; D043244:Ribonuclease III
keywords: Escherichia coli; RNA degradation; RNA sequencing; RNA stability; RNase III