Abstract
Regulation of RNA turnover is of utmost importance for controlling the concentration of transcripts and consequently cellular protein levels. Among the processes controlling RNA decay, small noncoding regulatory RNAs (sRNAs) have recently emerged as major new players. In this chapter, we describe and discuss protocols that can be used to measure sRNA concentration in vivo and to assess sRNA decay rates in Gram-negative bacteria. Precisely, we focus our analyses on the Escherichia coli Gram-negative bacterium as a model. The information described in this chapter provides a guideline to help develop a protocol in order to assess these important parameters and to identify RNA-processing enzymes involved in sRNA degradation processes.
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Abbreviations
- ncRNA:
-
Noncoding RNA
- nt:
-
Nucleotide
- NTD/CTD:
-
N-terminal/C-terminal domain
- PAGE:
-
PolyAcrylamide gel electrophoresis
- PAP:
-
Poly(A) polymerase
- PNPase:
-
Polynucleotide phosphorylase
- RNAP:
-
RNA polymerase
- RNAse:
-
Ribonuclease
- sRNA:
-
Small RNA
- ss/ds:
-
Single/double stranded
- Tm :
-
Melting temperature
References
Hui MP, Foley PL, Belasco JG (2014) Messenger RNA degradation in bacterial cells. Annu Rev Genet 48:537–559
Gorna MW, Carpousis AJ, Luisi BF (2011) From conformational chaos to robust regulation: the structure and function of the multi-enzyme RNA degradosome. Q Rev Biophys 45:105–145
Taghbalout A, Yang Q, Arluison V (2014) The Escherichia coli RNA processing and degradation machinery is compartmentalized within an organized cellular network. Biochem J 458:11–22
Soper T, Mandin P, Majdalani N, Gottesman S, Woodson SA (2010) Positive regulation by small RNAs and the role of Hfq. Proc Natl Acad Sci U S A 107:9602–9607
Aiba H (2007) Mechanism of RNA silencing by Hfq-binding small RNAs. Curr Opin Microbiol 10:134–139
Vogel J, Luisi BF (2011) Hfq and its constellation of RNA. Nat Rev Microbiol 9:578–589
Ikeda Y, Yagi M, Morita T, Aiba H (2011) Hfq binding at RhlB-recognition region of RNase E is crucial for the rapid degradation of target mRNAs mediated by sRNAs in Escherichia coli. Mol Microbiol 79:419–432
Mohanty BK, Maples VF, Kushner SR (2004) The Sm-like protein Hfq regulates polyadenylation dependent mRNA decay in Escherichia coli. Mol Microbiol 54:905–920
Church GM, Gilbert W (1984) Genomic sequencing. Proc Natl Acad Sci U S A 81:1991–1995
Andrade JM, Pobre V, Matos AM, Arraiano CM (2012) The crucial role of PNPase in the degradation of small RNAs that are not associated with Hfq. RNA 18:844–855
Saramago M, Barria C, Dos Santos RF, Silva IJ, Pobre V, Domingues S, Andrade JM, Viegas SC, Arraiano CM (2014) The role of RNases in the regulation of small RNAs. Curr Opin Microbiol 18:105–115
Andrade JM, Arraiano CM (2008) PNPase is a key player in the regulation of small RNAs that control the expression of outer membrane proteins. RNA 14:543–551
Viegas SC, Arraiano CM (2008) Regulating the regulators: how ribonucleases dictate the rules in the control of small non-coding RNAs. RNA Biol 5:230–243
Masse E, Escorcia FE, Gottesman S (2003) Coupled degradation of a small regulatory RNA and its mRNA targets in Escherichia coli. Genes Dev 17:2374–2383
Viegas SC, Silva IJ, Saramago M, Domingues S, Arraiano CM (2011) Regulation of the small regulatory RNA MicA by ribonuclease III: a target-dependent pathway. Nucleic Acids Res 39:2918–2930
Marujo PE, Hajnsdorf E, Le Derout J, Andrade R, Arraiano CM, Regnier P (2000) RNase II removes the oligo(A) tails that destabilize the rpsO mRNA of Escherichia coli. RNA 6:1185–1193
Reichenbach B, Maes A, Kalamorz F, Hajnsdorf E, Gorke B (2008) The small RNA GlmY acts upstream of the sRNA GlmZ in the activation of glmS expression and is subject to regulation by polyadenylation in Escherichia coli. Nucleic Acids Res 36:2570–2580
Sledjeski DD, Whitman C, Zhang A (2001) HFq is necessary for regulation by the untranslated RNA DsrA. J Bacteriol 183:1997–2005
De Lay N, Schu DJ, Gottesman S (2013) Bacterial small RNA-based negative regulation: Hfq and its accomplices. J Biol Chem 288:7996–8003
Vanzo NF, Li YS, Py B, Blum E, Higgins CF, Raynal LC, Krisch HM, Carpousis AJ (1998) Ribonuclease E organizes the protein interactions in the Escherichia coli RNA degradosome. Genes Dev 12:2770–2781
Khemici V, Poljak L, Luisi BF, Carpousis AJ (2008) The RNase E of Escherichia coli is a membrane-binding protein. Mol Microbiol 70:799–813
Kuwano M, Ono M, Endo H, Hori K, Nakamura K, Hirota Y, Ohnishi Y (1977) Gene affecting longevity of messenger RNA: a mutant of Escherichia Coli with altered mRNA stability. Mol Gen Genet 154:279–285
Ono M, Kuwano M (1979) A conditional lethal mutation in an Escherichia Coli strain with a longer chemical lifetime of messenger RNA. J Mol Biol 129:343–357
McDowall KJ, Hernandez RG, Lin Chao S, Cohen SN (1993) The ams-1 and rne-3071 temperature-sensitive mutations in the ams gene are in close proximity to each other and cause substitutions within a domain that resembles a product of the Escherichia coli mre locus. J Bacteriol 175:4245–4249
Kido M, Yamanaka K, Mitani T, Niki H, Ogura T, Hiraga S (1996) RNase E polypeptides lacking a carboxyl-terminal half suppress a mukB mutation in Escherichia coli. J Bacteriol 178:3917–3925
Ow MC, Kushner SR (2002) Initiation of tRNA maturation by RNase E is essential for cell viability in E. coli. Genes Dev 16:1102–1115
Anupama K, Leela JK, Gowrishankar J (2011) Two pathways for RNase E action in Escherichia coli in vivo and bypass of its essentiality in mutants defective for Rho-dependent transcription termination. Mol Microbiol 82:1330–1348
Jain C, Belasco JG (1995) RNase E autoregulates its synthesis by controlling the degradation rate of its own mRNA in Escherichia Coli: unusual sensitivity of the rne transcript to RNase E activity. Genes Dev 9:84–96
Morita T, Maki K, Aiba H (2005) RNase E-based ribonucleoprotein complexes: mechanical basis of mRNA destabilization mediated by bacterial noncoding RNAs. Genes Dev 19:2176–2186
Morita T, Kawamoto H, Mizota T, Inada T, Aiba H (2004) Enolase in the RNA degradosome plays a crucial role in the rapid decay of glucose transporter mRNA in the response to phosphosugar stress in Escherichia coli. Mol Microbiol 54:1063–1075
Mackie GA (2013) RNase E: at the interface of bacterial RNA processing and decay. Nat Rev Microbiol 11:45–57
Garrey SM, Mackie GA (2011) Roles of the 5′-phosphate sensor domain in RNase E. Mol Microbiol 80:1613–1624
Regnier P, Hajnsdorf E (1991) Decay of mRNA encoding ribosomal protein S15 of Escherichia coli is initiated by an RNase E-dependent endonucleolytic cleavage that removes the 3′ stabilizing stem and loop structure. J Mol Biol 217:283–292
Donovan WP, Kushner SR (1986) Polynucleotide phosphorylase and ribonuclease II are required for cell viability and mRNA turnover in Escherichia coli K-12. Proc Natl Acad Sci U S A 83:120–124
Yancey SD, Kushner SR (1990) Isolation and characterization of a new temperature-sensitive polynucleotide phosphorylase mutation in Escherichia coli K-12. Biochimie 72:835–843
Andrade JM, Hajnsdorf E, Regnier P, Arraiano CM (2009) The poly(A)-dependent degradation pathway of rpsO mRNA is primarily mediated by RNase R. RNA 15:316–326
Rasmussen AA, Eriksen M, Gilany K, Udesen C, Franch T, Petersen C, Valentin-Hansen P (2005) Regulation of ompA mRNA stability: the role of a small regulatory RNA in growth phase-dependent control. Mol Microbiol 58:1421–1429
Okello JB, Rodriguez L, Poinar D, Bos K, Okwi AL, Bimenya GS, Sewankambo NK, Henry KR, Kuch M, Poinar HN (2010) Quantitative assessment of the sensitivity of various commercial reverse transcriptases based on armored HIV RNA. PLoS One 5:e13931
Stahlberg A, Kubista M, Pfaffl M (2004) Comparison of reverse transcriptases in gene expression analysis. Clin Chem 50:1678–1680
Plumbridge JA, Dondon J, Nakamura Y, Grunberg-Manago M (1985) Effect of NusA protein on expression of the nusA, infB operon in E. coli. Nucleic Acids Res 13:3371–3388
Hajnsdorf E, Steier O, Coscoy L, Teysset L, Regnier P (1994) Roles of RNase E, RNase II and PNPase in the degradation of the rpsO transcripts of Escherichia oli: stabilizing function of RNase II and evidence for efficient degradation in an ams pnp rnb mutant. EMBO J 13:3368–3377
Ziolkowska K, Derreumaux P, Folichon M, Pellegrini O, Regnier P, Boni IV, Hajnsdorf E (2006) Hfq variant with altered RNA binding functions. Nucleic Acids Res 34:709–720
Arraiano CM, Yancey SD, Kushner SR (1988) Stabilization of discrete mRNA breakdown products in ams pnp rnb multiple mutants of Escherichia oli K-12. J Bacteriol 170:4625–4633
Hajnsdorf E, Braun F, Haugel-Nielsen J, Regnier P (1995) Polyadenylylation destabilizes the rpsO mRNA of Escherichia coli. Proc Natl Acad Sci U S A 92:3973–3977
Nogueira T, de Smit M, Graffe M, Springer M (2001) The relationship between translational control and mRNA degradation for the Escherichia coli threonyl-tRNA synthetase gene. J Mol Biol 310:709–722
Masse E, Gottesman S (2002) A small RNA regulates the expression of genes involved in iron metabolism in Escherichia coli. Proc Natl Acad Sci U S A 99:4620–4625
Lu F, Taghbalout A (2013) Membrane association via an amino-terminal amphipathic helix is required for the cellular organization and function of RNase II. J Biol Chem 288:7241–7251
Acknowledgments
This work was supported by the CNRS, CEA, and University Paris Diderot. We are particularly grateful to Bastien Cayrol (INRA/CIRAD) for his help in preparing this manuscript, and to Richard Lease (Ohio State Univ.) and Daniele Joseleau-Petit (Univ. Paris Diderot) for critical reading of the manuscript.
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Busi, F., Arluison, V., Régnier, P. (2018). Absolute Regulatory Small Noncoding RNA Concentration and Decay Rates Measurements in Escherichia coli . In: Arluison, V., Valverde, C. (eds) Bacterial Regulatory RNA. Methods in Molecular Biology, vol 1737. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7634-8_14
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DOI: https://doi.org/10.1007/978-1-4939-7634-8_14
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