Increased Stability of<i>bmr3</i>mRNA Results in a Multidrug-Resistant Phenotype in<i>Bacillus subtilis</i>

Ohki, Reiko; Tateno, Kozue

doi:10.1128/jb.186.21.7450-7455.2004

Cited by 17 publications

(15 citation statements)

References 25 publications

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“…5A. The moderately structured and strongly structured sequences were patterned, respectively, after the wild-type and mutant forms of mdr (or bmr3) mRNA (29). The strongly structured sequence was shown to increase mdr mRNA stability at least fourfold.…”

Section: Resultsmentioning

confidence: 94%

Processing and Stability of Inducibly ExpressedrpsOmRNA Derivatives inBacillus subtilis

Yao

Bechhofer

2009

J Bacteriol

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The Bacillus subtilis rpsO gene specifies a small (388-nucleotide), monocistronic mRNA that encodes ribosomal protein S15. We showed earlier that rpsO mRNA decay intermediates accumulated to a high level in a strain lacking polynucleotide phosphorylase. Here, we used inducibly expressed derivatives of rpsO, encoding smaller RNAs that had the complex 5 region deleted, to study aspects of mRNA processing in B. subtilis. An IPTG (isopropyl-␤-D-thiogalactopyranoside)-inducible rpsO transcript that contained lac sequences at the 5 end, called lac-rpsO RNA, was shown to undergo processing to result in an RNA that was 24 nucleotides shorter than full length. Such processing was dependent on the presence of an accessible 5 terminus; a lac-rpsO RNA that contained a strong stem-loop at the 5 end was not processed and was extremely stable. Interestingly, this stability depended also on ribosome binding to a nearby Shine-Dalgarno sequence but was independent of downstream translation. Either RNase J1 or RNase J2 was capable of processing lac-rpsO RNA, demonstrating for the first time a particular in vivo processing event that could be catalyzed by both enzymes. Decay intermediates were detected in the pnpA strain only for a lac-rpsO RNA that was untranslated. Analysis of processing of an untranslated lac-rpsO RNA in the pnpA strain shortly after induction of transcription suggested that endonuclease cleavage at 3-proximal sites was an early step in turnover of mRNA.The rate of decay of an mRNA is an important component in determining the level of gene expression. Studies of the mechanism of mRNA decay in Escherichia coli have progressed based on a detailed knowledge of the ribonucleases involved in the process and the construction of RNase mutant strains. A similar level of understanding of the mechanism of mRNA decay has not been achieved for the model grampositive organism Bacillus subtilis. Earlier studies of a number of B. subtilis mRNAs, some of which were constitutively or inducibly stable, revealed that mRNA decay in B. subtilis initiates from the 5Ј end (9) and that polynucleotide phosphorylase (PNPase) (encoded by the pnpA gene) plays a major role in 3Ј-to-5Ј exonucleolytic degradation (15,30,42). More recently, the role of RNase J1 and RNase J2 ribonucleases (18) has become paramount. Of the two, only RNase J1 is essential, and reduced expression of RNase J1 results in an increase in global mRNA half-life, suggesting a general role for RNase J1 in initiation of decay. RNase J1 has been shown to be involved in decay and processing of a number of specific RNAs (3,7,14,18,44), and a recent study demonstrated the effect of reduced RNase J1 and/or lack of RNase J2 on hundreds of B. subtilis mRNAs (26). While the RNase J enzymes were initially purified on the basis of their endoribonuclease activity, it was shown subsequently that RNase J1 also has 5Ј-to-3Ј exoribonuclease activity (27), which is inhibited by a 5Ј triphosphate end (14,25).A model for mRNA turnover in B. subtilis involves RNase J1in two possible pathways...

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Section: Resultsmentioning

confidence: 94%

Processing and Stability of Inducibly ExpressedrpsOmRNA Derivatives inBacillus subtilis

Yao

Bechhofer

2009

J Bacteriol

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“…trp Leader RNA with a 5Ј Stabilizer-Experiments in our laboratory and others (31)(32)(33), as well as a survey of stable B. subtilis mRNAs (20), have indicated that a 5Ј-terminal stem loop structure should have a strong stabilizing effect. We predicted that addition of such a structure would stabilize greatly the extremely unstable D1 RNA.…”

Section: Rna Sequencementioning

confidence: 99%

Bacillus subtilis trp Leader RNA

Deikus

Bechhofer

2009

Journal of Biological Chemistry

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In the presence of ample tryptophan, transcription from the Bacillus subtilis trp operon promoter terminates to give a 140-nucleotide trp leader RNA. Turnover of trp leader RNA has been shown to depend on RNase J1 cleavage at a single-stranded, AUrich region just upstream of the 3 transcription terminator. The small size of trp leader RNA and its strong dependence on RNase J1 cleavage for decay make it a suitable substrate for analyzing the requirements for RNase J1 target site specificity. trp leader RNAs with nucleotide changes around the RNase J1 target site were more stable than wild-type trp leader RNA, showing that sequences on either side of the cleavage site contribute to RNase J1 recognition. An analysis of decay intermediates from these mutants suggested limited 3-to-5 exonuclease processing from the native 3 end. trp leader RNAs were designed that contained wild-type or mutant RNase J1 targets elsewhere on the molecule. The presence of an additional RNase J1 cleavage site resulted in faster RNA decay, depending on its location. Addition of a 5 tail containing 7 A residues caused destabilization of trp leader RNAs. Surprisingly, addition at the 5 end of a strong stem loop structure that is known to stabilize other RNAs did not result in a longer trp leader RNA half-life, suggesting that the RNase J1 cleavage site may be accessed directly. In the course of these experiments, we found evidence that polynucleotide phosphorylase processivity was inhibited by a GCGGCCGC sequence.Protective features of the 5Ј and 3Ј ends of prokaryotic mRNAs explain why these RNA molecules are not degraded immediately by the multiple ribonucleases that are present in a prokaryotic cell. The presence of a nucleoside triphosphate at the 5Ј end renders this end a poor substrate for 5Ј-to-3Ј exonucleolytic decay (1-3) or 5Ј end-dependent endonucleolytic activity (4 -6). The strong stem loop structure found at the 3Ј end of many prokaryotic mRNAs, which is the transcription terminator structure, is resistant to 3Ј-to-5Ј exonucleolytic decay (7,8). A general model for the initiation of mRNA decay in prokaryotes, which is based on numerous studies in Escherichia coli, is as follows: initiation of decay occurs by an endonucleolytic cleavage in the body of the message. Such cleavage generates an upstream fragment with an unprotected 3Ј end, which is a good substrate for 3Ј-to-5Ј exonucleases, and a downstream fragment with a monophosphate nucleoside 5Ј end, which is a good substrate for additional endonuclease cleavages (9 -11). The downstream fragment could also be a good substrate for a 5Ј-to-3Ј exonuclease activity; however, such an activity is not known to exist in E. coli.The major decay-initiating endonuclease in E. coli is believed to be RNase E, a 5Ј end-dependent endonuclease. That is, RNase E endonuclease activity is usually contingent on prior binding to the 5Ј terminus, after which the enzyme tracks or loops to its target cleavage site (5, 12). RNase E vastly prefers an RNA substrate with a monophosphate 5Ј end (4, 5, 13)...

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“…(B) Sequence and predicted structure of the 5ЈSS mRNA stabilizer. on April 30, 2019 by guest http://jb.asm.org/ mol) and has been shown in other contexts to function as a 5Ј stabilizer (13,18). The addition of the 5ЈSS to ⌬ermC mRNA substantially increased the half-life in the wild-type strain to 18.3 min ( Table 1).…”

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confidence: 82%

Decay of a Model mRNA in Bacillus subtilis by a Combination of RNase J1 5′ Exonuclease and RNase Y Endonuclease Activities

et al. 2011

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The involvement of the recently characterized 5 exonuclease activity of RNase J1 and endonuclease activity of RNase Y in the turnover of ⌬ermC mRNA in Bacillus subtilis was investigated. Evidence is presented that both of these activities determine the half-life of ⌬ermC mRNA.The 260-nucleotide (nt) ⌬ermC mRNA ( Fig. 1A) has served as a useful model for determining the effects of structural and translational elements on mRNA stability in Bacillus subtilis (1,16,17,20). The involvement of the essential RNase J1, a bifunctional enzyme that has both endonuclease and 5Ј exonuclease activities (10, 12), in ⌬ermC mRNA decay was reported previously (20): the half-life of ⌬ermC mRNA increased 2.5-fold, from 7.5 min to 18.5 min, in an RNase J1-limited strain. The terms "RNase J1-" and "RNase Y-limited strain" refer to strains in which expression of the particular RNase is under p spac promoter control, which results in an RNase concentration severalfold lower than that in the wildtype strain (7). A 130-nt derivative of ⌬ermC mRNA, which had an in-frame deletion of nt 87 to 215 and which we call here ⌬ermC-del (Fig. 1A), showed a 2-fold increase in half-life in the wild-type strain (14.6 min). This result indicated strongly that exonucleolytic degradation by RNase J1 from the 5Ј end could not account completely for ⌬ermC mRNA turnover, as this mode of decay was unlikely to be affected by an internal deletion. We hypothesized that one or more RNase J1 endonuclease cleavage sites were located between nt 87 and 215 and that deletion of this sequence increased half-life by eliminating these decay initiation sites. Whether exonucleolytic degradation by RNase J1 from the 5Ј end also contributed to determining the half-life of the native transcript was not investigated. At the time of this study, we were unaware of the existence of RNase Y, a recently discovered essential endoribonuclease (5, 15) that may be even more important than RNase J1 for turnover of bulk mRNA (15). Here, we address the roles of RNase J1 5Ј exonuclease activity and RNase Y endonuclease activity in ⌬ermC mRNA decay.The effect of RNase Y on ⌬ermC mRNA half-life was determined in an RNase Y-limited strain (15). Northern blot analysis, performed as described previously (19), showed that reduction of RNase Y levels resulted in an almost 2-fold increase in ⌬ermC mRNA half-life (7.5 min to 13.8 min; Table 1). Thus, in addition to RNase J1, RNase Y is also involved in determining ⌬ermC mRNA stability.We next measured the effect of RNase Y on the half-life of ⌬ermC-del mRNA and found that it was unchanged from that of the wild-type strain (14.6 min and 15.0 min; Table 1). This suggested that the deleted sequence (nt 87 to 215) in ⌬ermC-del mRNA contained one or more RNase Y cleavage sites; deletion of this sequence gave an mRNA that was no longer targeted by RNase Y.We showed previously that prominent ⌬ermC mRNA decay intermediates of Ͻ140 nt could be detected with a 3Ј-proximal probe in the RNase J1-limited strain, although not in the wild-type strain (20). We h...

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Increased Stability ofbmr3mRNA Results in a Multidrug-Resistant Phenotype inBacillus subtilis

Cited by 17 publications

References 25 publications

Processing and Stability of Inducibly ExpressedrpsOmRNA Derivatives inBacillus subtilis

Processing and Stability of Inducibly ExpressedrpsOmRNA Derivatives inBacillus subtilis

Bacillus subtilis trp Leader RNA

Decay of a Model mRNA in Bacillus subtilis by a Combination of RNase J1 5′ Exonuclease and RNase Y Endonuclease Activities

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