rnb, ?em class="a-plus-plus">rnr and ?em class="a-plus-plus">pnp). We compared each of the mutant transcriptome with the wild-type to determine the global effects of the deletion of each exoribonucleases in exponential phase. We determined that the deletion of RNase II significantly affected 187 transcripts, while deletion of RNase R affects 202 transcripts and deletion of PNPase affected 226 transcripts. Surprisingly, many of the transcripts are actually down-regulated in the exoribonuclease mutants when compared to the wild-type control. The results obtained from the transcriptomic analysis pointed to the fact that these enzymes were changing the expression of genes related with flagellum assembly, motility and biofilm formation. The three exoribonucleases affected some stable RNAs, but PNPase was the main exoribonuclease affecting this class of RNAs. We confirmed by qPCR some fold-change values obtained from the RNA-Seq data, we also observed that all the exoribonuclease mutants were significantly less motile than the wild-type cells. Additionally, RNase II and RNase R mutants were shown to produce more biofilm than the wild-type control while the PNPase mutant did not form biofilms. Conclusions In this work we demonstrate how deep sequencing can be used to discover new and relevant functions of the exoribonucleases. We were able to obtain valuable information about the transcripts affected by each of the exoribonucleases and compare the roles of the three enzymes. Our results show that the three exoribonucleases affect cell motility and biofilm formation that are two very important factors for cell survival, especially for pathogenic cells." />
Next generation sequencing analysis reveals that the ribonucleases RNase II, RNase R and PNPase affect bacterial motility and biofilm formation in E. coli
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  • 作者：Vania Pobre ; Cecília M Arraiano
  • 关键词：Exoribonucleases ; RNase II ; RNase R ; PNPase ; Transcriptome ; RNA ; Seq ; Motility ; Biofilm formation
  • 刊名：BMC Genomics
  • 出版年：2015
  • 出版时间：December 2015
  • 年：2015
  • 卷：16
  • 期：1
  • 全文大小：1,095 KB
  • 参考文献：1. Andrade JM, Pobre V, Silva IJ, Domingues S, Arraiano CM. The role of 3'-5' exoribonucleases in RNA degradation. Prog Mol Biol Transl Sci. 2009;85:187-29. CrossRef
    2. Arraiano CM, Andrade JM, Domingues S, Guinote IB, Malecki M, Matos RG, et al. The critical role of RNA processing and degradation in the control of gene expression. FEMS Microbiol Rev. 2010;34(5):883-23.
    3. Cannistraro VJ, Kennell D. The reaction mechanism of ribonuclease II and its interaction with nucleic acid secondary structures. Biochem et Biophys Acta. 1999;1433:170-7.
    4. Spickler C, Mackie A. Action of RNases II and Polynucleotide Phosphorylase against RNAs containing stem-loops of defined structure. J Bacteriol. 2000;182:2422-. CrossRef
    5. Coburn GA, Mackie GA. Overexpression, purification, and properties of / Escherichia coli ribonuclease II. J Biol Chem. 1996;271(2):1048-3. CrossRef
    6. Folichon M, Marujo PE, Arluison V, Le Derout J, Pellegrini O, Hajnsdorf E, et al. Fate of mRNA extremities generated by intrinsic termination: detailed analysis of reactions catalyzed by ribonuclease II and poly(A) polymerase. Biochimie. 2005;87(9-0):819-6. CrossRef
    7. Hajnsdorf E, Steier O, Coscoy L, Teysset L, Régnier P. Roles of RNase E, RNase II and PNPase in the degradation of the / rpsO transcripts of / Escherichia coli: stabilizing function of RNase II and evidence for efficient degradation in an / ams pnp rnb mutant. EMBO J. 1994;13(14):3368-7.
    8. Marujo PE, Hajnsdorf E, Le Derout J, Andrade R, Arraiano CM, Régnier P. RNase II removes the oligo(A) tails that destabilize the / rpsO mRNA of / Escherichia coli. RNA. 2000;6(8):1185-3. CrossRef
    9. Mohanty BK, Kushner SR. Polynucleotide phosphorylase, RNase II and RNase E play different roles in the / in vivo modulation of polyadenylation in / Escherichia coli. Mol Microbiol. 2000;36(4):982-4. CrossRef
    10. Pepe CM, Maslesa-Galic S, Simons RW. Decay of the IS10 antisense RNA by 3' exoribonucleases: evidence that RNase II stabilizes RNA-OUT against PNPase attack. Mol Microbiol. 1994;13(6):1133-2. CrossRef
    11. Cheng ZF, Deutscher MP. Purification and characterization of the / Escherichia coli exoribonuclease RNase R. Comparison RNase II J Biol Chem. 2002;277(24):21624-.
    12. Vincent HA, Deutscher MP. Substrate recognition and catalysis by the exoribonuclease RNase R. J Biol Chem. 2006;281(40):29769-5. CrossRef
    13. Andrade JM, Hajnsdorf E, Régnier P, Arraiano CM. The poly(A)-dependent degradation pathway of / rpsO mRNA is primarily mediated by RNase R. RNA. 2009;15(2):316-6. CrossRef
    14. Awano N, Rajagopal V, Arbing M, Patel S, Hunt J, Inouye M, et al. / Escherichia coli RNase R has dual activities, helicase and RNase. J Bacteriol. 2010;192(5):1344-2. CrossRef
    15. Cheng ZF, Deutscher MP. Quality control of ribosomal RNA mediated by polynucleotide phosphorylase and RNase R. Proc Natl Acad Sci U S A. 2003;100(11):6388-3. CrossRef
    16. Cairr?o F, Cruz A, Mori H, Arraiano CM. Cold shock induction of RNase R and its role in the maturation of the quality control mediator SsrA/tmRNA. Mol Microbiol. 2003;50(4):1349-0. CrossRef
    17. Andrade JM, Cairr?o F, Arraiano CM. RNase R affects gene expression in stationary phase: regulation of / ompA. Mol Microbiol. 2006;60(1):219-8. CrossRef
    18. Chen C, Deutscher MP. RNase R is a highly unstabl
  • 刊物主题：Life Sciences, general; Microarrays; Proteomics; Animal Genetics and Genomics; Microbial Genetics and Genomics; Plant Genetics & Genomics;
  • 出版者：BioMed Central
  • ISSN：1471-2164
文摘
Background The RNA steady-state levels in the cell are a balance between synthesis and degradation rates. Although transcription is important, RNA processing and turnover are also key factors in the regulation of gene expression. In Escherichia coli there are three main exoribonucleases (RNase II, RNase R and PNPase) involved in RNA degradation. Although there are many studies about these exoribonucleases not much is known about their global effect in the transcriptome. Results In order to study the effects of the exoribonucleases on the transcriptome, we sequenced the total RNA (RNA-Seq) from wild-type cells and from mutants for each of the exoribonucleases (?em class="a-plus-plus">rnb, ?em class="a-plus-plus">rnr and ?em class="a-plus-plus">pnp). We compared each of the mutant transcriptome with the wild-type to determine the global effects of the deletion of each exoribonucleases in exponential phase. We determined that the deletion of RNase II significantly affected 187 transcripts, while deletion of RNase R affects 202 transcripts and deletion of PNPase affected 226 transcripts. Surprisingly, many of the transcripts are actually down-regulated in the exoribonuclease mutants when compared to the wild-type control. The results obtained from the transcriptomic analysis pointed to the fact that these enzymes were changing the expression of genes related with flagellum assembly, motility and biofilm formation. The three exoribonucleases affected some stable RNAs, but PNPase was the main exoribonuclease affecting this class of RNAs. We confirmed by qPCR some fold-change values obtained from the RNA-Seq data, we also observed that all the exoribonuclease mutants were significantly less motile than the wild-type cells. Additionally, RNase II and RNase R mutants were shown to produce more biofilm than the wild-type control while the PNPase mutant did not form biofilms. Conclusions In this work we demonstrate how deep sequencing can be used to discover new and relevant functions of the exoribonucleases. We were able to obtain valuable information about the transcripts affected by each of the exoribonucleases and compare the roles of the three enzymes. Our results show that the three exoribonucleases affect cell motility and biofilm formation that are two very important factors for cell survival, especially for pathogenic cells.
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