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Knockdown or silencing of a specific gene presents a powerful strategy for elucidating gene function in a variety of organisms. To date, efficient silencing methods have been established in eukaryotes, but not bacteria. In this chapter, an efficient and versatile gene silencing method using artificial small RNA (afsRNA) is described. For this purpose, target-recognizing sequences were introduced in specially designed RNA scaffolds to exist as single-stranded stretches in afsRNA. The translation initiation region of target genes was used as the sequence for afsRNA recognition, based on the theory that this site is usually highly accessible to ribosomes, and therefore, possibly, afsRNA. Two genes transcribed as monocistrons were tested with our protocol. Both genes were effectively silenced by their cognate afsRNAs.

Most small noncoding RNAs (sRNAs) are known to base pair with target mRNAs and regulate mRNA stability or translation to trigger various changes in the cell metabolism of Escherichia coli. The SdsR sRNA is expressed specifically during the stationary phase and represses tolC and mutS expression. However, it was not previously known whether the growth-phase-dependent regulation of SdsR is important for cell growth. Here, we ectopically expressed SdsR during the exponential phase and examined cell growth and survival. We found that ectopic expression of SdsR led to a significant and Hfq-dependent cell death with accompanying cell filamentation. This SdsR-driven cell death was alleviated by overexpression of RyeA, an sRNA transcribed on the opposite DNA strand, suggesting that SdsR/RyeA is a novel type of toxin-antitoxin (T/A) system in which both the toxin and the antitoxin are sRNAs. We defined the minimal region required for the SdsR-driven cell death. We also performed RNA-seq analysis and identified 209 genes whose expression levels were altered by more than two-fold following pulse expression of ectopic SdsR at exponential phase. Finally, we found that that the observed SdsR-driven cell death was mainly caused by the SdsR-mediated repression of yhcB, which encodes an inner membrane protein.

E-mail: Younghoon.Lee@kaist.ac.krReceived January 28, 2014, Accepted March 18, 2014Key Words : E. coli, Thermosensitive, dbpA knockout, ribE, Keio collectionAll Escherichia coli genes are classified into three groups,specifically, genes essential for cell growth (essential),dispensable for cell growth (non-essential), and unknown(essential or non-essential). Basically, essential genes arecritical for survival of an organism and more highly con-served in evolution. On the other hand, a gene in a growingstrain with a null-type mutation is classified as non-essential.

ssrS-encoded 6S RNA is an abundant noncoding RNA that binds σ(70)-RNA polymerase and regulates expression at a subset of promoters in Escherichia coli. It is transcribed from two tandem promoters, ssrS P1 and ssrS P2. Regulation of transcription from two ssrS promoters in 6S RNA biogenesis was examined. Both P1 and P2 were growth phase-dependently regulated. Depletion of 6S RNA had no effect on growth-phase-dependent transcription from either promoter, whereas overexpression of 6S RNA increased P1 transcription and decreased P2 transcription, suggesting that transcription from P1 and P2 is subject to feedback activation and feedback inhibition, respectively. This feedback regulation disappeared in Δfis strains, supporting involvement of Fis in this process. The differential feedback regulation may provide a means for maintaining appropriate cellular concentrations of 6S RNA.

The long noncoding RNA BC200 (brain cytoplasmic RNA, 200 nucleotides) acts as a translational modulator of local protein synthesis at dendrites. BC200 RNA has been shown to inhibit translation in vitro, but it remains unknown how this translation inhibition might be controlled in a cell. Here, we performed yeast three-hybrid screening and identified hnRNP E1 and hnRNP E2 as BC200 RNA-interacting proteins. We found that: these hnRNA proteins could restore BC200 RNA-inhibited translation; BC200 RNA interacts with hnRNP E1 and E2 mainly through its unique 3' C-rich domain; and the RNA binding specificities and modes of the two proteins differed somewhat. Our results offer new insights into the regulation of BC200 RNA-mediated translation inhibition.

Figure 1. Effects on sequestration of C5 protein on M1 RNA stability in endoribonuclease-deficient cells. Total cellular RNA was isolated from strains GW10 (wild type), GW11 (rng‒), and GW20 (rne) containing plasmids at the indicated times after the rifampicin treatment. Each RNA sample (30 μg) was fractionated on a 5% polyacrylamide gel containing 7 M urea and analyzed by northern blotting. A stem P12-specific probe was used as a probe for M1 RNA. Escherichia coli RNase P was initially characterized as a tRNA-processing enzyme that removes extraneous 5' sequences from precursor tRNAs to generate mature 5' termini. In addition to tRNA processing, RNase P is involved in the processing of other non-tRNA substrates (e.g., 4.5S RNA and tmRNA) and the decay of several mRNAs.. The RNase P holoenzyme is composed of two subunits, a large RNA subunit (M1 RNA, 377 nucleotides) and a small basic protein (C5 protein, 119 amino acids). M1 RNA is the catalytic subunit that can perform the reaction as a ribozyme in the absence of C5 protein in vitro, but both the protein and RNA components are essential in vivo. C5 protein stabilizes the catalytically active conformation of M1 RNA and modulates its substrate specificity. Previously, we have shown that the turnover rate of free M1 RNA increases when C5 protein is sequestered by truncated M1 RNA transcripts, such as P12-deleted M1 RNA, in the cell, suggesting that the protein also functions as a metabolic stabilizer of M1 RNA. The size of C5 protein is only one-tenth of M1 RNA. Therefore, it is an interesting issue how this small protein can protect such a large RNA molecule from degradation. To address this issue, in this study, we tried to identify RNases responsible for the rapid turnover of free M1 RNA. For this purpose, we introduced into cells plasmid pLMd12, which overproduced P12deleted M1 RNA derivative as a C5 protein-interacting RNA, so that this plasmid-borne, overexpressed M1 RNA derivative could sequester C5 protein available for M1 RNA binding and thereby generate a protein-free form of M1 RNA within cells. Generally RNA degradation is initiated by endoribonucleolytic cleavage and RNase E plays an important role in this endoribonucleolytic cleavage. Therefore, we investigated the turnover rate of free M1 RNA in strains GW20 and GW11, which showed a temperature-sensitive RNase E phenotype and a RNase G mutant phenotype, respectively. Total RNAs prepared from GW20 and GW11 cells containing plasmid pLMdP12 were analyzed. The cellular levels of M1 RNA level after the treatment of rifampicin that inhibits transcription were monitored by northern blot analysis and then the metabolic stability of M1 RNA was assessed (Fig. 1). We observed no decrease of the M1 RNA stability in both cells. Surprisingly the M1 RNA stability was not decreased in the wild type control strain GW10 either. This contrasts our previous report that the sequestration of C5 protein via overexpression of C5 protein-interacting RNA, such as P12-deleted M1 RNA, reduces the metabolic stability

Five Sib antitoxin RNAs, members of a family of cis-encoded small regulatory RNAs (sRNAs) in Escherichia coli, repress their target mRNAs, which encode Ibs toxins. This target repression occurs only between cognate sRNA–mRNA pairs with an exception of ibsA. We performed co-transformation assays to assess the ability of SibC derivatives to repress ibsC expression, thereby revealing the regions of SibC that are essential for ibsC mRNA recognition. SibC has two target recognition domains, TRD1 and TRD2, which function independently. The target site for TRD1 is located within the ORF of ibsC, whereas the target site for TRD2 is located in the translational initiation region. The TRD1 sequence is sufficient to repress ibsC expression. In contrast, TRD2 requires a specific structure in addition to the recognition sequence. An in vitro structural probing analysis showed that the initial interactions at these two recognition sites allowed base-pairing to progress into the flanking sequences. Displacement of the TRD1 and TRD2 domains of SibC by the corresponding domains of SibD changed the target specificity of SibC from ibsC to ibsD, suggesting that these two elements modulate the cognate target recognition of each Sib RNA by discriminating among non-cognate ibs mRNAs.

Bacterial small RNAs (sRNAs) are known regulators in many physiological processes. In Escherichia coli, a large number of sRNAs have been predicted, among which only about a hundred are experimentally validated. Despite considerable research, the majority of their functions remain uncovered. Therefore, collective analysis of the roles of sRNAs in specific cellular processes may provide an effective approach to identify their functions. Here, we constructed a collection of plasmids overexpressing 99 individual sRNAs and analyzed their effects on biofilm formation and related phenotypes. Thirty-three sRNAs significantly affecting these cellular processes were identified. No consistent correlations were observed, except that all five sRNAs suppressing type I fimbriae inhibited biofilm formation. Interestingly, IS118, yet to be characterized, suppressed all the processes. Our data not only reveal potentially critical functions of individual sRNAs in biofilm formation and other phenotypes but also highlight the unexpected complexity of sRNA-mediated metabolic pathways leading to these processes.

Knockdown or silencing of a specific gene presents a powerful strategy for elucidating gene function in a variety of organisms. To date, efficient silencing methods have been established in eukaryotes, but not bacteria. In this chapter, an efficient and versatile gene silencing method using artificial small RNA (afsRNA) is described. For this purpose, target-recognizing sequences were introduced in specially designed RNA scaffolds to exist as single-stranded stretches in afsRNA. The translation initiation region of target genes was used as the sequence for afsRNA recognition, based on the theory that this site is usually highly accessible to ribosomes, and therefore, possibly, afsRNA. Two genes transcribed as monocistrons were tested with our protocol. Both genes were effectively silenced by their cognate afsRNAs.

IbsC is a small toxin protein in Escherichia coli, whose expression is repressed by a cis-acting small noncoding RNA, SibC (renamed from RygC or QUAD1c). Overexpression of IbsC or the absence of SibC transcription induces the expression of both psp operon (pspABCDE) and pspG gene encoding phage shock proteins, whose expression were known to be induced by multiple environment stresses or agents such as filamentous phage infection, osmotic shock, continued incubation in stationary phase, heat shock, and ethanol treatment. Although exogenous overexpression of IbsC leads to cell death, physiological roles of IbsC remain unknown especially because the strain without ibsC gene in the chromosome shows no growth defects. Considering that only Psp proteins are induced by expression of IbsC, not by other similar small toxin proteins such as LdrD, ShoB, and TisB, the role of IbsC might specifically be related to phage shock proteins. The psp operon is transcribed from pspA promoter by RNA polymerase holoenzyme containing the alternative stress-responsive sigma factor 54 (σ). Transcription from pspA promoter requires PspF as an activator protein that binds to the upstream region of the core promoter elements. Furthermore, PspA inhibits the σ-dependent pspA transcription by interacting with PspF and exerting its negative effects on transcription activation by PspF. Since the IbsC expression causes the induction of pspA transcription, it is attempting to see how the signal of IbsC gets transduced into the transcription activation. The mechanism involved in linking IbsC to pspA transcription is essential for understanding not only the physiological functions of IbsC, but also the regulation mechanism of transcription from σdependent promoters in response to environmental stresses. To understand the molecular mechanism of the pspA induction by IbsC, it is necessary to know which factors are involved in the activation of pspA transcription under conditions of IbsC expression. Although an in vitro transcription system for pspA promoter analysis was previously set up, it is difficult to identify in vitro transcripts because their transcription termination sites are unclear. To overcome the drawback of the previous in vitro transcription system, in this study, we constructed plasmid pPR56 containing a transcription fusion of pspA promoter and rnpB terminator by replacing the rnpB promoter-containing DNA fragment of pLMd23-wt with the pspA promoter-containing fragment. The pspA promoter-containing fragment spanning from −320 through +56 of pspA, which also includes two UAS sequences (UAS I and UAS II) as PspF-binding sites and the IHF binding site, was subcloned into the BamHI/ EcoRI linearlized pLMd23-wt not having the rnpB promotercontaining DNA fragment, to generate the fusion plasmid pPR56 (Fig. 1). The rnpB terminator region in the fusion construct contained the rnpB sequence from +331 to +1286, which includes the three rnpB terminators T1, T2, and T3 leading to transcription termination at +413, +526, and +638, respectively. Therefore, this pPR56 construct was designed to generate pspA-rnpB fusion transcripts of 146 nt terminating at T1 (pspAT1), 259 nt at T2 (pspAT2), and 371 nt at T3 (pspAT3) if transcription starts at the transcription initiation site of pspA. Using supercoiled plasmid pPR56 DNA as a template, in vitro transcription was carried out by adding Eσ and PspF. The pspAT1 transcript of 145 nt was produced as a major band although minor pspAT2 and pspAT3 products were also observed (Fig. 2). The increased abundance of three transcripts was observed with the incremental amount of PspF protein, while RNA I of 108 nt (a transcript transcribed from its own σ specific promoter of the plasmid DNA) was not produced. In contrast, the same transcription reaction performed with Eσ did not produce the pspAT transcripts. Instead, this reaction generated RNA I. All our data confirmed that the pspA transcripts were produced from the σ-specific pspA promoter of pPR56. To

SummaryLIN28-mediated processing of the microRNA (miRNA) let-7 has emerged as a multilevel program that controls self-renewal in embryonic stem cells. LIN28A is believed to act primarily in the cytoplasm together with TUT4/7 to prevent final maturation of let-7 by Dicer, whereas LIN28B has been suggested to preferentially act on nuclear processing of let-7. Here, we find that SET7/9 monomethylation in a putative nucleolar localization region of LIN28A increases its nuclear retention and protein stability. In the nucleoli of human embryonic stem cells, methylated LIN28A sequesters pri-let-7 and blocks its processing independently of TUT4/7. The nuclear form of LIN28A regulates transcriptional changes in MYC-pathway targets, thereby maintaining stemness programs and inhibiting expression of early lineage-specific markers. These findings provide insight into the molecular mechanism underlying the posttranslational methylation of nuclear LIN28A and its ability to modulate pluripotency by repressing let-7 miRNA expression in human embryonic stem cells.

Objectives To systematically analyse the interplay between the expression of Hfq-associated small non-coding RNAs (sRNAs) and antibiotic susceptibility in Gram-negative bacteria. Methods To identify the roles of sRNAs in the antibiotic susceptibility of Escherichia coli and Salmonella species, susceptibility tests, growth analyses and viability assays were performed using E. coli Hfq-associated sRNAs from overexpression libraries. Prediction, susceptibility testing of gene knockouts and expression analysis of target genes under conditions of sRNA overexpression or knockout were performed to identify candidate targets for modulating antibiotic susceptibility. Results The susceptibilities of E. coli strains overexpressing each of the 26 known Hfq-dependent sRNAs to major classes of antibiotics were determined. Induced expression of 17 sRNAs modulated the susceptibility of E. coli to antibiotics. Among them, four sRNA knockout strains partially or completely reversed susceptibility phenotypes of sRNA overexpression. The phenotype of OxyS, RseX or MicF was not entirely dependent on the presence of Hfq protein, in contrast to the dependency of previously characterized roles. The function of eight of nine sRNAs was found to be conserved in the response to antibiotics in Salmonella. Some MicF- or RyeB-mediated cellular target genes and pathways that may be important for the regulation of antibiotic susceptibility were identified. Finally, the overexpression of RyeB potentiated the efficacy of levofloxacin against MDR strains. Conclusions Our data indicate that Hfq-associated sRNAs potentially enable bacteria to adapt to antibiotic challenges via multifaceted approaches. Therefore, sRNA-based applications will form a new antibiotic arsenal for combating the rise in antibiotic resistance.

A simple, rapid, and sensitive electrophoretic mobility shift assay (EMSA) can be successfully used to analyze RNA-RNA interactions. The EMSA of RNA-RNA complexes can be further used to evaluate the specificity of interactions using competitor RNAs in combination with their mutated versions or nonspecific RNAs, such as yeast tRNA. RNA is simply prepared by in vitro transcription from PCR product templates. Detailed experimental descriptions for EMSA-based analysis of specific RNA-RNA interactions between Sib RNAs and ibs mRNAs as a representative example are presented.

A simple, rapid, and sensitive electrophoretic mobility shift assay (EMSA) can be successfully used to analyze RNA-RNA interactions. The EMSA of RNA-RNA complexes can be further used to evaluate the specificity of interactions using competitor RNAs in combination with their mutated versions or nonspecific RNAs, such as yeast tRNA. RNA is simply prepared by in vitro transcription from PCR product templates. Detailed experimental descriptions for EMSA-based analysis of specific RNA-RNA interactions between Sib RNAs and ibs mRNAs as a representative example are presented.

Escherichia coli and related enteric bacteria can survive under extreme acid stress condition at least for several hours. RpoS is a key factor for acid stress management in many enterobacteria. Although three rpoS-activating sRNAs, DsrA, RprA, and ArcZ, have been identified in E. coli, it remains unclear how these small RNA molecules participate in pathways leading to acid resistance (AR). Here, we showed that overexpression of ArcZ, DsrA, or RprA enhances AR in a RpoS-dependent manner. Mutant strains with deletion of any of three sRNA genes showed lowered AR, and deleting all three sRNA genes led to more severe defects in protecting against acid stress. Overexpression of any of the three sRNAs fully rescued the acid tolerance defects of the mutant strain lacking all three genes, suggesting that all three sRNAs perform the same function in activating RpoS required for AR. Notably, acid stress led to the induction of DsrA and RprA but not ArcZ.

An artificial small RNA (afsRNA) scaffold was designed from an Escherichia coli sRNA, SibC. Using the lacZ reporter system, the gene silencing effects of afsRNAs were examined to explore the sRNA-mediated gene-silencing mechanisms in E. coli. Substitution of the original target recognition sequence with a new sequence recognizing lacZ mRNA led to effective reduction of lacZ gene expression. Single-strandedness of the target recognition sequences in the scaffold was essential for effective gene silencing. The target recognition sequence was shortened to 10 nt without significant loss of gene silencing, although this minimal length was limited to a specific target mRNA sequence. In cases where afsRNAs had mismatched (forming internal loops) or unmatched (forming bulges) regions in the middle of the target recognition sequence, internal loop-forming afsRNAs were more effective in gene silencing than those that formed bulges. Unexpectedly, gene silencing by afsRNA was not decreased but increased on hfq disruption in E. coli, particularly when interactions between afsRNA and mRNA were weak, suggesting that Hfq is possibly involved in destabilization of the RNA–RNA duplex, rather than enhancement of base pairing.

Cnu (an OriC-binding nucleoid protein) associates with H-NS. A variant of Cnu was identified as a key factor for filamentous growth of a wild-type Escherichia coli strain at 37 degrees C. This variant (CnuK9E) bears a substitution of a lysine to glutamic acid, causing a charge reversal in the first helix. The temperature-dependent filamentous growth of E. coli bearing CnuK9E could be reversed by either lowering the temperature to 25 degrees C or lowering the CnuK9E concentration in the cell. Gene expression analysis suggested that downregulation of dicA by CnuK9E causes a burst of dicB transcription, which, in turn, elicits filamentous growth. In vivo assays indicated that DicA transcriptionally activates its own gene, by binding to its operator in a temperature-dependent manner. The antagonizing effect of CnuK9E with H-NS on DNA-binding activity of DicA was stronger at 37 degrees C, presumably due to the lower operator binding of DicA at 37 degrees C. These data suggest that the temperature-dependent negative effect of CnuK9E on DicA binding plays a major role in filamentous growth. The C-terminus of DicA shows significant amino acid sequence similarity to the DNA-binding domains of RovA and SlyA, regulators of pathogenic genes in Yersinia and Salmonella, respectively, which also show better DNA-binding activity at 25 degrees C.

The yeast three-hybrid system (Y3H), a powerful method for identifying RNA-binding proteins, still suffers from many false positives, due mostly to RNA-independent interactions. In this study, we attempted to efficiently identify false positives by introducing a tetracycline operator (tetO) motif into the RPR1 promoter of an RNA hybrid expression vector. We successfully developed a tight tetracycline-regulatable RPR1 promoter variant containing a single tetO motif between the transcription start site and the A-box sequence of the RPR1 promoter. Expression from this tetracycline-regulatable RPR1 promoter in the presence of tetracycline-response transcription activator (tTA) was positively controlled by doxycycline (Dox), a derivative of tetracycline. This on-off control runs opposite to the general knowledge that Dox negatively regulates tTA. This positively controlled RPR1 promoter system can therefore efficiently eliminate RNA-independent false positives commonly observed in the Y3H system by directly monitoring RNA hybrid expression.