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Nucleic Acids Research Advance Access published online on May 27, 2009
Nucleic Acids Research, doi:10.1093/nar/gkp447
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© 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods online

Specific gene silencing by artificial trans-encoded small noncoding RNAs in bacteria

Rubin Cheng, Cuicui Miao, Qianhong Gong, Yuchao Gu, Xinzhi Lu, Feng Han and Wengong Yu*

Department of Molecular Biology, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, People's Republic of China

*To whom correspondence should be addressed. Tel: +86 532 82031680; Fax: +86 532 82033054; Email: yuwg66{at}ouc.edu.cn

Received February 16, 2009. Revised May 9, 2009. Accepted May 12, 2009.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY DATA
 FUNDING
 REFERENCES
 
Recently, numerous small noncoding RNAs (sRNAs) with important regulatory roles have been identified in bacteria. As their eukaryotic counterparts, a major class of bacterial trans-encoded sRNAs, acts by basepairing with target mRNAs, resulting in changes in translation and stability of the mRNA. RNA interference (RNAi) has become an extraordinarily powerful RNA silencing tool for elucidating and manipulating gene functions in eukaryotes. However, such an effective RNA silencing tool remains to be developed for prokaryotes. In this study, we described firstly the use of artificial trans-encoded sRNAs (atsRNAs) for specific gene silencing in bacteria. Based on the common structural characteristics of natural bacterial trans-encoded sRNAs, we developed the designing principle of atsRNA. Most of the atsRNAs effectively suppressed the expression of exogenous EGFP gene and endogenous uidA gene in Escherichia coli. Further studies demonstrated that the mRNA base-pairing region and AU rich Hfq binding site were crucial for the activity of atsRNA. The atsRNA-mediated gene silencing was Hfq dependent. atsRNA led to translational repression and RNase-E-dependent degradation of target mRNA, and the translation inhibition was the primary event for gene silencing. Our findings demonstrated that atsRNA was an effective RNA tool for specific gene silencing in bacteria.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY DATA
 FUNDING
 REFERENCES
 
The small noncoding RNAs (sRNAs) have attracted great interest as ubiquitous regulators in all kingdoms of life. The eukaryotic microRNA (miRNA) and short-interfering RNA (siRNA) have been making a splash during the past few years. Recently, with the development of the experimental and computational approaches, hundreds of sRNAs have been identified in bacteria, especially in Escherichia coli (1,2). The bacterial sRNAs constitute a structurally diverse class of molecules that are typically of 50–250 nt in length and do not contain expressed open reading frames (ORFs). They have been shown to be involved in many cellular processes in prokaryotes, including translational quality control, protein inhibition, iron homeostasis, outer membrane protein biogenesis, sugar metabolism, quorum sensing, survival in stationary phase and virulence regulation of pathogens (3–8). Those sRNAs whose functions have been characterized can be sorted into three general categories: (i) sRNAs that have intrinsic catalytic activity or are components of ribonucleoproteins, (ii) sRNAs that affect protein activity by structurally mimicking other nucleic acids and (iii) sRNAs that regulate gene expression by basepairing with target mRNAs, changing the translation and stability of the mRNA. sRNAs in the latter category appear to be the best characterized and most abundant in bacteria. Most of these identified sRNAs are trans-encoded, located at chromosomal positions different from their targets. The majority of these trans-encoded sRNAs, such as DicF, MicF, OxyS, Spot42, RyhB and GcvB, repress the translation of target mRNAs (4,9–13).

A large class of trans-encoded sRNAs silences their target mRNAs by binding tightly to Hfq, a highly abundant RNA chaperone protein that also binds the target mRNA in a number of cases studied (14–16). Basepairing between these sRNAs and their target mRNAs requires Hfq which is proposed to enhance the stability of sRNAs in vivo, by protecting them from degradation. Hfq has also been shown to interact with proteins like poly(A) polymerase I (PAP I), polynucleotide phosphorylase (PNP) and RNase E which are involved in mRNA decay and it is shown to form fibres in vitro, the physiological significance of which is unknown (17–21).

The small RNAs in eukaryotes, such as miRNA and siRNA, have become a powerful experimental technique for silencing gene expression both in cultured cells and living organisms (22–24). However, such an effective RNA-silencing tool remains to be developed for prokaryotes in which the main methods of deciphering gene function are still homologous recombination and transposon mutagenesis. Additionally, the application of traditional antisense RNA (asRNA) is also limited due to its low efficacy (25,26). Therefore, effective and convenient RNA-silencing methods are urgently needed for prokaryotic organisms. Recently, the emergence of widespread trans-encoded sRNAs that act as regulators of gene expression by basepairing with their targets in bacteria provide us a promising perspective for gene function investigation.

In the present study, we described firstly the use of artificial trans-encoded sRNAs (atsRNAs) for specific gene silencing in bacteria. A series of atsRNAs targeting specific genes were designed and synthesized, and most of them significantly suppressed the expression of target genes in an Hfq-dependent manner. Our results demonstrated that atsRNA was an efficient approach for specific gene silencing in bacteria.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 SUPPLEMENTARY DATA
 FUNDING
 REFERENCES
 
Media and growth conditions
Bacteria were grown aerobically in LB medium. Antibiotics were used at the following concentrations: ampicillin, 100 µg/ml; kanamycin, 30 µg/ml; tetracycline, 20 µg/ml; and chloramphenicol, 20 µg/ml. Bacterial growth was monitored according to the optical density (OD) at 600 nm.

Strains and plasmids
Escherichia coli MC4100 (F– araD139 {Delta}(argF-lac) U169 rpsL150 relA1 deoC1 rbsR fthD5301 fruA25 {lambda}–) (a gift from R. Gary Sawers) and CSH26 (F– ara {Delta}(lac-pro) thi) (a gift from Masaaki Wachi) were used as the parent wild-type strains. The hfq mutant strain GSO81 (MC4100 hfq-1::{Omega}) was a gift from Gisela Storz; the temperature-sensitive rne-1 mutant strain HAT103 (CSH26 zce-726::Tn10 rne-1) was a gift from Masaaki Wachi. EGFP gene was inserted into pET-24a (+) (Novagen, USA), yielding recombinant plasmid pET24a-EGFP. A constitutive promoter P1 was cloned from plasmid pBR322 and inserted into pET24a-EGFP to drive the transcription of EGFP gene. The fragment of P1 promoter, EGFP gene and T7 transcriptional terminator were amplified and inserted into pACYC177 (New England Biolabs, USA), creating recombinant plasmid pACYC-P1-EGFP. The pACYC-P1-EGFP was a low-copy vector carrying a P15A origin of replication, kanamycin resistance gene and a constitutive P1 promoter.

Constructs for atsRNA biosynthesis
The mRNA base-pairing regions of atsRNAs were designed according to the 5' UTR of target exogenous EGFP gene and endogenous uidA gene (U00096 [GenBank] ). The other two modules (Hfq binding site and Rho-independent terminator) were extracted from well-known natural Hfq-dependent trans-encoded sRNAs. The three elements were selected by simple random sampling techniques and assembled into a series of atsRNA candidates from which atsRNA was chosen based on the predicted secondary structure by MFOLD (27). Then two complementary DNA oligonucleotides for atsRNA biosynthesis were synthesized by Shanghai Sangon, and then annealed in a 20 µl reaction mixture (containing 10 mM Tris–HCl, pH 8.0, 50 mM NaCl and 1 mM EDTA). The annealing mixture was heated at 95°C for 5 min and then transferred to a 70°C water bath and slowly cooled to room temperature. After ligation, the double-stranded DNA was double digested with EcoRI/HindIII and inserted into the corresponding sites of pRI (a gift from Gisela Storz), creating plasmid recombinants of atsRNA expression vector. atsRNA expression vector was a high-copy vector compatible with pACYC-P1-EGFP and carrying a pBR322 origin of replication and a bla ampicillin resistance gene. The transcription of atsRNA gene would precisely start at the putative +1 site of atsRNA under the control of tac promoter (28). The sequences of synthesized atsRNA genes are listed in Supplementary Table S1.

Fluorescence measurement
Escherichia coli strains were cultured at 30°C and the isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM to induce the expression of atsRNA genes when OD600 of the culture reached 0.5. To measure the fluorescence, bacterial cells were collected after induction for 30 min by centrifugation at 5000 g and 4°C for 5 min, washed and re-suspended in 500 µl of phosphate-buffered saline (50 mM, pH 7.0). Cells were disrupted with Vcx750 watt ultrasonic processor (Sonics and Materials, Inc., USA) at 0°C and 9 kHz for 2 min, and then the cell free extract was obtained by centrifugation at 15 000 g and 4°C for 15 min. The protein concentration was assayed with BCA Protein Assay kit (Pierce, USA). As much as 300 µg of proteins were transferred to a 96-well microtitre plate (BD Falcon, USA) with the final volume adjusted with phosphate-buffered saline to 200 µl. Fluorescence was measured using SpectraMax M5 (Molecular devices, USA) with excitation filter at 474 nm and emission filter at 515 nm. Three independent sets of experiments were performed.

Activity assay of beta-glucuronidase
The activity of beta-glucuronidase was assayed using the 4-methyl-umbelliferyl-β-D-glucuronide (4-MUG) as substrate (29,30). Bacterial strain was cultured at 30°C with IPTG added to a final concentration of 1 mM when OD600 reached 0.5. Bacterial cells were collected after induction for 30 min by centrifugation at 5000 g and 4°C for 5 min. The cells were washed and resuspended in GUS assay buffer. Cells were ultrasonically disrupted at 0°C and 9 kHz for 2 min, and the supernatant was obtained by centrifugation at 15 000 g and 4°C for 15 min. The protein concentration was assayed as was done for fluorescence measurement. As much as 300 µg of proteins were added into a new tube with final volume adjusted with GUS assay buffer to 400 µl. One hundred microliters of 5 mM MUG was transferred to each tube. After reaction at 37°C for 1 h, 50 µl of the mixture was pipetted into a new tube, mixed with 900 µl of stop buffer. The fluorescence was detected using SpectraMax M5 with excitation filter at 388 nm and emission filter at 480 nm. The activity of beta-glucuronidase was indicated directly with the fluorescence value. Three independent sets of experiments were performed.

Northern blot analysis
Bacterial total RNA was isolated using Trizol reagent (Invitrogen, USA) according to the manufacturer's instructions. Digoxigenin-labeled RNA probes against atsRNAs, uidA mRNA, 5S rRNA and 16S rRNA were obtained from the specific PCR products containing a T7 promoter by in vitro transcription using the DIG-Labeling kit (Roche Diagnostics, Germany). The primer sequences used for northern blotting were as follows: forward primer of atsRNA genes, 5'-GTTGACAATTAATCATCGGCT-3', reverse primer of atsRNA genes, 5'-CTAATACGACTCACTATAGGGAGCTCTCATCCGCCAAAACAG-3'; forward primer of uidA gene, 5'-TATACGCCATTTGAAGCC-3', reverse primer of uidA gene, 5'-CTAATACGACTCACTATAGGGAGAAGCCAGTAAAGTAGAACG-3'; forward primer of 5S rRNA gene, 5'-GAATTTGCCTGGCGGCAGTAGCGCG-3', reverse primer of 5S rRNA gene, 5'-CTAATACGACTCACTATAGGGAGATGCCTGGCAGTTCCCTACTCTCGC-3'; forward primer of 16S rRNA gene, 5'-TGGCGGACGGGTGAGTAATG-3', reverse primer of 16S rRNA gene, 5'-CTAATACGACTCACTATAGGGAGGGCTGCTGGCACGGAGTTAG-3'. For northern blot analysis of atsRNA, 5 µg of total RNA was separated on 7 M urea/TBE/8% polyacrylamide gels and subsequently transferred to a positively charged nylon membrane (Hybond N+, Pharmacia, USA) by electroblotting in 0.5x TBE at 15 V for 1 h. For northern blot analysis of uidA mRNA, 5 µg of total RNA was separated by electrophoresis on 1.5% agarose gels containing formaldehyde. The RNA in agarose gels was then transferred overnight to the positively charged nylon membranes by capillary action in 20x SSC. Probe hybridization and detection were carried out according to the supplier's instruction (DIG RNA Labeling kit, Roche Diagnostics, Germany). For stability experiments, transcription initiation was inhibited by adding rifampicin to growing cells to a final concentration of 500 µg/ml (time 0). Northern blots were performed as above except that rifampicin was added.

Electrophoretic mobility shift assays
The hfq gene without stop coden was amplified from MC4100 and inserted into the BamHI/XhoI sites of pET-24a, creating recombinant plasmid pET24a-hfq. The His-tagged Hfq protein was overproduced from BL21 (DE3) strain and purified by Ni2+-NTA column (Qiagen, Germany). Digoxigenin-labeled atsRNAs were obtained from specific PCR products containing a T7 promoter by in vitro transcription using the DIG-RNA-Labeling kit (Roche Diagnostics). The primer sequences used were as follows: forward primer of atsRNA genes, 5'-CTAATACGACTCACTATAGGGAGGTTGACAATTAATCATCGGCT-3', reverse primer of atsRNA genes, 5'-CTCTCATCCGCCAAAACAG-3'. Electrophoretic mobility shift assay (EMSA) reactions were carried out at 37°C for 30 min. Labeled atsRNAs (10 nM) were incubated without or with increasing amounts of purified Hfq protein in a 20 µl reaction in binding buffer (10 mM Tris–HCl, pH 7.4, 5% glycerol (v/v), 5 mM magnesium acetate, 40 mM KCl, 1.0 mM dithiothreitol, 67 ng/µl yeast tRNA, 1.5 µg/µl heparin, 2% β-mercaptoethanol). RNA–protein complexes were subsequently separated on native 6% polyacrylamide gels and transferred onto a positively charged nylon membrane (Hybond N+, Pharmacia). Then the Dig-labeled RNA–protein complexes were detected according to the supplier's instruction (Roche Diagnostics, Germany).

Quantitative real-time polymerase chain reaction
Bacterial total RNA was isolated using RNeasy Midi Kit (Qiagen, Germany) and cDNA was synthesized using High Capacity cDNA Archival Kit (Applied Biosystems, USA) following manufacturer's protocols, respectively. Primers were designed using Primer Express (Applied Biosystems, USA) and synthesized by Shanghai Sangon. The primer sequences used for QPCR were as follows: forward primer of EGFP gene, 5'-CGGCATGGACGAGCTGTAC-3', reverse primer of EGFP gene, 5'-GCTTCCTTTCGGGCTTTGT-3'; forward primer of uidA gene, 5'-TGAGCGTCGCAGAACATTACA-3', reverse primer of uidA gene, 5'-GCCACTGGCGGAAGCAA-3'; forward primer of 16S rRNA gene, 5'-TGACGCTCAGGTGCGAAAG-3', reverse primer of 16S rRNA gene, 5'-CAAGGGCACAACCTCCAAGT-3'. The reaction volume was 10 µl consisting of 1x SYBR Green PCR Master Mix (Applied Biosystems, USA), 200 nM of each gene-specific forward primer and reverse primer and 0.02 ng of cDNA template. ROX was used to correct the fluorescence signals and 16S rRNA gene was used as the normalizer for each sample. Reactions were run in a 7500 Real-Time PCR system (Applied Biosystems, USA) under standard reaction conditions: 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Three independent sets of experiments were performed.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 SUPPLEMENTARY DATA
 FUNDING
 REFERENCES
 
Designing principle of atsRNA
The previous studies have shown that natural trans-encoded sRNAs that act through basepairing require three basic elements, a region that base-pairs with target mRNA, an Hfq binding site and the Rho-independent terminator (1,2,31–33). Therefore, we firstly analyzed the sequences of well-known bacterial trans-encoded sRNAs, such as Spot42 (4,14), RyhB (5,18), MicF (9,34), OxyS (11,15,35), DsrA (36,37) and Qrr1 (7,38), and divided them into three modules that were referred to as mRNA basepairing region, Hfq binding site and Rho-independent terminator in this study (Supplementary Figure S1). The mRNA base-pairing region of the sRNA was imperfectly complementary with the 5' untranslated region (5' UTR) of the target mRNA, especially the Shine-Dalgarno (SD) sequences. Although the Hfq binding sites of natural sRNAs shared no homology, the common characteristic of these sequences was rich in AU nucleotides. The Rho-independent terminators typically shared common structural features, which consisted of a GC-rich inverted repeat followed by a run of U residues. Next, the secondary structures of these well-known natural sRNAs were predicted by MFOLD program (27), and then their common characteristics were analyzed. These sRNAs were highly structured, forming at least two stem–loops by mRNA base-pairing region and Rho-independent terminator, respectively. In general, the Hfq binding site located at the AU-rich area between these two stem–loops in the predicted secondary structure.

Based on the common structural characteristics of natural bacterial trans-encoded sRNAs, we developed the principle and process for atsRNA design (Figure 1). (i) atsRNA should be a modular structure consisting of three elements: mRNA base-pairing region, Hfq binding site and Rho-independent terminator. (ii) The mRNA base-pairing region (20–30 nt in length) should be designed according to the 5' UTR (or part of the coding regions) of target mRNA, especially the SD sequence, and then the sequence composition should be adjusted appropriately to make it form a stem–loop structure. (iii) The Hfq binding sites and Rho-independent terminators were extracted from the well-studied endogenous bacterial trans-encoded sRNAs, such as RyhB, Spot42, DsrA and OxyS. The Hfq binding sites generally corresponded to a 12–19-nt AU-rich region. (iv) The three component parts were selected randomly and then assembled into a series of atsRNA candidates according to the combination order listed in Figure 1. Then their secondary structures were predicted by MFOLD program. (v) atsRNAs should be selected from atsRNA candidates whose predicted secondary structures meet the following criteria: atsRNAs should form two to four stem–loops, including one formed by mRNA base-pairing region and one formed by Rho-independent terminator. The Hfq binding site should locate between two stem–loops. Additionally, the start codon AUG should be avoided in atsRNA in order to prevent the translation of atsRNA. (vi) The selected atsRNAs were synthesized, cloned into the expression vector and transformed into host bacteria. Then, the efficacies of these atsRNAs were determined.


Figure 1
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  		Figure 1. Schematic representation of the designing process for atsRNA.

 
Designing and characterization of atsRNAs targeting against specific genes
To evaluate the feasibility of this method, the exogenous EGFP gene (enhanced green fluorescent protein) was selected as the target. The EGFP expression vector was constructed based on pACYC177 plasmid, and referred to as pACYC-P1-EGFP which was a low-copy vector carrying a P15A origin of replication, kanamycin resistance gene and a constitutive P1 promoter. The EGFP gene was transcribed under the control of the constitutive P1 promoter. According to the above designing principle, six atsRNAs (GY1–GY6) targeting against EGFP gene were designed and synthesized (Table 1 and Supplementary Table S1), then inserted into the expression vector pRI (28), a high-copy vector compatible with pACYC-P1-EGFP. The pACYC-P1-EGFP and pRI carrying atsRNA genes were co-transformed into E. coli MC4100. As shown in Figure 2A, GY2 and GY6 significantly suppressed the expression of EGFP gene (P < 0.01); and GY1, GY4 and GY5 did so slightly (P < 0.05); while GY3 almost had no repressing effect (P < 0.1). To further test the application of this method, 10 atsRNAs (CY1–CY10) targeting against endogenous uidA gene that encodes beta-glucuronidase were designed and synthesized (Table 1 and Supplementary Table S1). Plasmid pRI carrying these atsRNA genes were transformed into E. coli MC4100, respectively, and the expression level of uidA gene was measured by detecting the activity of beta-glucuronidase. As shown in Figure 2B, CY1, CY4, CY6 and CY9 suppressed the expression of uidA gene obviously (P < 0.01); and CY3, CY5, CY7 and CY10 did so slightly (P < 0.05); while CY2 and CY8 almost had no effect (P < 0.05). The most effective atsRNA CY9 triggered a 72% reduction in expression of target gene. To determine whether the repression of target genes was indeed due to the expression of atsRNAs, we then analyzed the transcripts of atsRNAs by northern blotting. As shown in Figure 2C and D, atsRNAs were transcribed with the predicted length, indicating that the reduction on target gene was due to the expression of atsRNA. Furthermore, to confirm whether the expression of atsRNAs interfere with the physiology of host bacteria, we first detected the growth curve of host bacteria after the induction of atsRNA. As shown in Supplementary Figure S2, the expression of atsRNAs had no effect on growth rate of host bacteria. Then we swapped atsRNA expression plasmids (CY series for GY series and vice versa) as a control of specificity. As shown in Supplementary Figure S3, the expression level of EGFP and uidA gene had no reduction after the induction of atsRNA. These results demonstrated that atsRNA was effective in suppressing the expression of specific bacterial genes.


Figure 2
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  		Figure 2. Effect of atsRNAs on gene silencing in E. coli. (A) The effect of atsRNAs on target EGFP gene. (B) The effect of atsRNAs on target uidA gene. (C) Northern blot analysis of atsRNA transcripts targeting EGFP gene. (D) Northern blot analysis of atsRNA transcripts targeting uidA gene. In (A) and (B), the expression of target gene was measured after the induction of atsRNA with 1 mM IPTG for 30 min. Bacterial cells containing the empty vector pRI were used as the control. Average values were from triplicate experiments with std values indicated. In (C) and (D), total RNAs were purified from cells after the expression of atsRNAs were induced with 1 mM IPTG for 30 min and analyzed using probes specific for atsRNAs respectively. The positions of RNA size standards are indicated on the left. The 5S rRNA was used as the loading control.

 

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  		Table 1. Summary of atsRNAs in this study

 
Structural and functional relationship of atsRNA
To gain more information for designing atsRNAs, the sequences and predicted structures of those effective and noneffective atsRNAs were compared. From the primary sequences of atsRNAs, there were no obvious differences in full length of the effective and noneffective atsRNAs (Table 1 and Supplementary Table S1). The mRNA base-pairing regions of those atsRNAs imperfectly paired with the 5' UTR of the target mRNAs according to the predicted results by LALIGN (Supplementary Figure S4 and Table S1). This result indicted that atsRNA acted by basepairing with target mRNA. The complementary sequences between atsRNAs and their targets ranged mainly from 19 to 23 nt in length, and there were no obvious differences in sequence composition between them (Supplementary Figure S4 and Table S1). Since the putative interacting sequences were maintained in all atsRNAs (effective and noneffective), we presumed that the different effect of these atsRNAs was due to the secondary structure or stability or the other two component parts of atsRNA.

In the predicted secondary structure, all the effective atsRNAs showed two or three stem–loop structures and putative AU-rich Hfq binding sites locating between two stem–loops (Supplementary Figure S5). Their mRNA base-pairing regions formed a stem–loop structure, while two atsRNA candidates without stem–loop structure in mRNA base-pairing region were noneffective. To confirm the role of stem–loop structure, the mRNA base-pairing regions of CY4 and CY6 were adjusted appropriately to make them lost the stem–loop in predicted secondary structure, yielding atsRNA mutants L-CY4 and L-CY6, respectively (Figure 3A). The complementary sequences of L-CY4 and L-CY6 were similar to that of CY4 and CY6 (Supplementary Figure S6) and the Hfq binding site and Rho-independent terminators remained constant in the mutant construct, respectively (Supplementary Table S2). As shown in Figure 3A, L-CY4 and L-CY6 lost repressing effect on the target gene (P < 0.01). Northern blotting results showed that the amounts of L-CY4 and L-CY6 decreased compared to that of the corresponding atsRNAs (Supplementary Figure S7). However, the reduced amounts of atsRNA mutants could not counteract the loss of repressing effect on target gene. After normalization of the interference efficiency of each atsRNAs to relative RNA amounts, the relative interference efficiency of L-CY4 and L-CY6 decreased dramatically (Supplementary Figure S7). This result suggested that the lost activity of mutants L-CY4 and L-CY6 was due to the loss of stem–loop structure but not the reduced stability. These results demonstrated that the stem–loop structure formed by mRNA base-pairing region was necessary for the activity of atsRNA.


Figure 3
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  		Figure 3. Secondary structure and function of atsRNA. (A) The predicted secondary structures and effect of atsRNA mutants L-CY4 and L-CY6 designed based on CY4 and CY6, respectively. Compared with the corresponding atsRNAs, L-CY4 and L-CY6 lost the stem–loop of mRNA base-pairing region in secondary structure. (B) The predicted secondary structures and effect of atsRNA mutants designed based on CY9. CY9-L1, CY9-L2, CY9-L3 and CY9-L4 were CY9 mutants with different number of unpaired nucleotides in the loop structure of mRNA base-pairing region. In (A) and (B), the Hfq binding site and Rho-independent terminator remained constant during the mutation. The sequences of predicted pairing region (indicated with arrows) and Hfq binding site (indicated with asterisks) of atsRNA are marked. The secondary structure of atsRNA is predicted at 37°C by MFOLD program. The activity of β-glucuronidase was assayed after the expression of atsRNA was induced with 1 mM IPTG for 30 min. Bacterial cells containing empty vector pRI were used as the control. Average values were from triplicate experiments with std values indicated.

 
The initial interaction between endogenous sRNA and mRNA was a stretch of unpaired nucleotides in a loop structure of sRNA in some cases (14,15,18,39). The length of the unpaired nucleotides in the loop formed by mRNA base-pairing region of CY9 was 9 nt. To determine the optimal length of unpaired nucleotides in the loop, the number of unpaired nucleotides of CY9 was changed appropriately, yielding atsRNA mutants CY9-L1 (3 nt in the loop), CY9-L2 (5 nt in the loop), CY9-L3 (7 nt in the loop) and CY9-L4 (13 nt in the loop). All of the mutants were maintained similar secondary structure with CY9 (Figure 3B). In addition, the complementary sequences of these mutants were similar to that of CY9 (Supplementary Figure S8) and the Hfq binding site and Rho-independent terminators remained constant in these mutant constructs (Supplementary Table S2). As shown in Figure 3B, the interference efficiency of CY9-L3 achieved 85%, while the interference efficiency of CY9-L1, CY9-L2 and CY9-L4 was lower than that of CY9 (P < 0.01). Furthermore, the relative interference efficiency of CY9-L3 was much higher than that of other atsRNAs (Supplementary Figure S9). This result demonstrated that the length of unpaired nucleotides in the loop structure formed by mRNA base-pairing region was important for the functioning of atsRNA and the optimal length of unpaired nucleotides was 7 for CY9.

The secondary structure of CY1 was similar to that of CY9 (Figure 4A); however, its interference efficiency was lower than that of CY9 (Figure 2B). The minimum free energy of CY1 (–16.9 kcal/mol) was higher than that of CY9 (–21.8 kcal/mol), indicating that it was easier to be degraded. To verify the predicted results, the stability of CY1 and CY9 was determined by northern blotting. As shown in Figure 4A, the half-life of CY9 was much longer than that of CY1. Furthermore, to increase the stability of CY1, atsRNA mutant N-CY1 was constructed by adding a 5' stem–loop structure without pairing sequence with target mRNA (Figure 4B). The mutant N-CY1 had the same mRNA base-pairing region, Hfq binding site and Rho-independent terminator with that of CY1 (Supplementary Table S2). As expected, the predicted minimum free energy of N-CY1 was reduced to –20.7 kcal/mol and the stability of N-CY1 was improved according to the northern blotting results (Supplementary Figure S10). The interference efficiency was increased compared with that of CY1 (P < 0.05) (Figure 4B). Additionally, the relative interference efficiency of N-CY1 was also slightly higher than that of CY1 (Supplementary Figure S10). These results demonstrated that the lower interference efficiency of CY1 was due to its unstable structure and the stability of atsRNA was crucial for its activity.


Figure 4
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  		Figure 4. Stability and function of atsRNA. (A) The predicted secondary structure and stability of atsRNAs CY1 and CY9. The expression of atsRNAs was induced with 1 mM IPTG for 30 min, and then rifampicin was added. The incubation was continued further and cells were harvested at the indicated time points for RNA preparation. Total RNA was analyzed using probes specific for CY1, CY9 and 5S rRNA, respectively. (B) The predicted secondary structure and repressing effect of CY1 and N-CY1. N-CY1 was constructed by adding a 5' stem–loop structure without pairing sequence with target mRNA. It held same mRNA base-pairing region, Hfq binding site and Rho-independent terminator with CY1. The activity of beta-glucuronidase was assayed after the expression of atsRNA was induced with 1 mM IPTG for 30 min. Bacterial cells containing empty vector pRI were used as the control. Average values were from triplicate experiments with std values indicated. In (A) and (B), the sequences of predicted pairing region (indicated with arrows) and Hfq binding site (indicated with asterisks) of atsRNA are marked. The secondary structure of atsRNA is predicted at 37°C by MFOLD program.

 
The mRNA base-pairing region and Hfq binding site are crucial for the activity of atsRNA
To confirm the effect of each component of atsRNA, a series of atsRNA mutants was designed. Since the secondary structure and stability played important roles in the functioning of atsRNA, these mutants should be maintained the original structure and predicted minimum free energy to the most extent.

First, the mRNA base-pairing regions of GY2 and CY9 were replaced by nonpairing sequences with target mRNA, respectively, yielding B-P-null-GY2 and B-P-null-CY9 (Figure 5A and B). The two mutants held similar secondary structure and minimum free energy with the corresponding atsRNAs. And the Hfq binding site and Rho-independent terminator remained constant during these mutations (Supplementary Table S3). Northern blotting results showed that these mutations did not alter the stability of the two corresponding atsRNAs (Supplementary Figure S11). As shown in Figure 5A and B, the two mutants lost the repressing effect on target genes (P < 0.05), demonstrating that mRNA base-pairing region was necessary for the functioning of atsRNA. This result also suggested that atsRNA acted by pairing with target mRNA. Second, the AU-rich Hfq binding sites of GY2 and CY9 were replaced by GC-rich sequences, respectively, yielding Hfq-null-GY2 and Hfq-null-CY9 with similar secondary structure and minimum free energy to the corresponding atsRNAs (Figure 5A and B). Similarly, the mRNA base-pairing region and Rho-independent terminator remained constant during this mutation (Supplementary Table S3). It was found that the interference efficiency of Hfq-null-GY2 and Hfq-null-CY9 was attenuated dramatically in comparison with that of GY2 and CY9 (P < 0.05). Then, the stability of mutants Hfq-null-GY2 and Hfq-null-CY9 were determined by northern blotting analysis (Supplementary Figure S12). Interestingly, the half-lives of the two mutants were much shorter than that of the corresponding atsRNAs. The reduced stability of mutants was presumed to be due to the reduced ability to bind Hfq. To confirm whether the decreased repressing effect was due to the reduced RNA amounts or not, we normalized the interference efficiency of each atsRNAs to relative RNA amounts. As shown in Supplementary Figure S12, the relative interference efficiency of the two mutants decreased certainly compared with that of the corresponding atsRNAs. This result indicated that the decreased interference efficiency was due to the lost Hfq binding site but not due to the reduced RNA amounts in vivo. It demonstrated that Hfq binding site was crucial for the stability and functioning of atsRNA.


Figure 5
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  		Figure 5. Effect of mRNA basepairing region and Hfq binding site on the functioning of atsRNA. (A) The predicted secondary structures and effect of atsRNA mutants B-P-null-GY2 and Hfq-null-GY2 designed based on GY2. (B) The predicted secondary structures and effect of atsRNA mutants B-P-null-CY9 and Hfq-null-CY9 designed based on CY9. In (A) and (B), the sequences of predicted pairing region (indicated by arrows) and Hfq binding site (indicated by asterisks) of atsRNA are marked. The secondary structure was predicted at 37°C by MFOLD program. Compared with the corresponding atsRNAs, the mRNA base-pairing regions of B-P-null-GY2 and B-P-null-CY9 were non-pairing sequences with target mRNAs and the Hfq binding sites of Hfq-null-GY2 and Hfq-null-CY9 were GC rich sequences. Furthermore, the two other modules of atsRNA remained constant during this mutation. The expression of target gene was measured after the induction of atsRNA with 1 mM IPTG for 30 min. Bacterial cells containing empty vector pRI were used as the control. Average values were from triplicate experiments with std values indicated.

 
The atsRNA-mediated gene silencing is Hfq dependent
To further confirm the role of Hfq, the EGFP expression vector pACYC-P1-EGFP and effective atsRNAs, GY1, GY2 and GY6, were first co-transformed into E. coli MC4100 and its hfq mutant strain GSO81 (15), respectively. These atsRNAs significantly suppressed the expression of EGFP gene in MC4100; however, they lost the repressing effects in GSO81 (P < 0.01) (Figure 6A). In addition, atsRNAs CY4, CY6 and CY9 were able to suppress the expression of target gene uidA in MC4100 but not in GSO81 (P < 0.01) (Figure 6B). Furthermore, the interference efficiencies of these atsRNAs were recovered after a multicopy plasmid containing hfq gene was transformed into the mutant strain GSO81 (Supplementary Figure S13). It is worth noting that the expression of target gene uidA decreased dramatically in GSO81 compared to the wild-type strain MC4100 in the absence of atsRNAs (Figure 6B). The importance of Hfq in cellular physiology has been acknowledged when the broadly pleiotropic phenotypes of an E. coli hfq mutant were characterized (40). Deletion of hfq could lead to the increase on oxidation of carbon sources. In this study, the target gene uidA encodes beta-glucuronidase, which is involved in the degradation of carbon compounds. Hfq was hypothesized to have effect on utilization of carbon sources and therefore affect the expression of uidA gene. Nonetheless, these findings demonstrated that the atsRNA-mediated gene silencing was Hfq dependent.


Figure 6
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  		Figure 6. Effect of Hfq on atsRNA-mediated gene silencing. (A) The effect of atsRNA GY1, GY2 and GY6 on EGFP gene in strains MC4100 and its hfq mutant GSO81. (B) The effect of atsRNA CY4, CY6 and CY9 on uidA gene in strains MC4100 and its hfq mutant GSO81. (C) The stability of atsRNA GY2 in MC4100 and its hfq mutant GSO81. (D) The stability of atsRNA CY9 in MC4100 and its hfq mutant GSO81. In (A) and (B), bacteria cells containing empty vector pRI were used as the control. The expression of target gene was measured after the induction of atsRNA with 1 mM IPTG for 30 min. Average values were from triplicate experiments with std values indicated. In (C) and (D), the expression of atsRNAs was induced with 1 mM IPTG for 30 min, and then rifampicin was added. The incubation was continued further and cells were harvested at the indicated time points for RNA preparation. Total RNA was analyzed using probes specific for GY2, CY9 and 5S rRNA, respectively.

 
Given that Hfq is important for the stability of natural trans-encoded sRNAs (14,41), we then determined the stability of atsRNAs GY2 and CY9 in isogenic hfq+ MC4100 and its hfq mutant strain GSO81. As shown in Figure 6C and D, the atsRNAs was very stable in MC4100, whereas it was rapidly degraded in the hfq mutant. This result demonstrated that Hfq was required for atsRNA stability. The effect of Hfq on atsRNA stability and function suggested that Hfq is binding to atsRNAs. To validate this conclusion, we determined the binding affinity of Hfq for atsRNAs GY2, CY9 and their corresponding Hfq binding site null mutants by EMSA in vitro (Supplementary Figure S14). Hfq was found to bind with high affinity to atsRNA GY2 and CY9, while it was defective in binding to Hfq-null-GY2 and Hfq-null-CY9. This result demonstrated that Hfq does bind with atsRNA and the Hfq binding site of atsRNA is crucial for the binding affinity. Thereafter, we detected whether Hfq could facilitate the interaction between atsRNA and target mRNA in vitro. As shown in Supplementary Figure S15, Hfq strongly enhanced the complex formation between atsRNA and target mRNA, indicating that Hfq could also facilitate the binding between atsRNA and target.

atsRNA leads to translational repression and RNase-E-dependent degradation of target mRNA
As described above, atsRNAs led to translational repression of target gene (Figures 2 and 5). Here, we tested whether atsRNA caused the degradation of target mRNAs. As shown in Figure 7A and B, the amount of target mRNAs decreased obviously after the induction of atsRNA by quantitative real-time polymerase chain reaction (QPCR). Furthermore, the northern blotting result showed that the half-life of uidA gene was reduced dramatically after the induction of CY9 (Figure 7C). These results demonstrated that atsRNAs led to the destabilization of target mRNA. To further elucidate the mechanism of atsRNA-mediated gene silencing, atsRNAs CY4 and CY9 was transformed into E. coli K12 strain CSH26 and its temperature-sensitive rne-1 mutant strain HAT103 (42), respectively. As shown in Figure 8A, CY4 and CY9 significantly suppressed the expression of uidA gene in both CSH26 and HAT103 strains at 30°C (P < 0.01). The two atsRNA still repressed the expression of target gene in HAT103 at 42°C (P < 0.005), indicating that the atsRNA-mediated gene silencing was RNase E independent (Figure 8A). The amount of uidA mRNA decreased obviously after the induction of atsRNAs in both CSH26 and its rne-1 mutant strain HAT103 at 30°C (Figure 8B), whereas the amount of uidA mRNA had no changes in HAT103 at 42°C (Figure 8C). Furthermore, the two atsRNAs could lead to degradation of target uidA mRNA in HAT103 even when it was cultured at 42°C after a multicopy plasmid containing rne gene was transformed into the mutant strain (Supplementary Figure S16). These results demonstrated that atsRNA led to an RNase-E-dependent degradation of target mRNA. Taken together, atsRNA downregulated target mRNA expression primarily by inhibiting translation, and that the RNase-E-dependent degradation of target mRNA was not necessary for gene silencing.


Figure 7
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  		Figure 7. atsRNA leads to the degradation of target mRNA. (A) Quantitative real-time PCR analysis of EGFP mRNA in MC4100. (B) Quantitative real-time PCR analysis of uidA mRNA in MC4100. In (A) and (B), the mRNA level was detected after the expression of atsRNA was induced with 1 mM IPTG for 30 min. Bacterial cells containing empty vector pRI were used as the control. The value was normalized to the level of 16S rRNA in each sample. (C) Northern blot analysis of stability of uidA mRNA in cells containing empty vector pRI and CY9. The expression of atsRNAs was induced with 1 mM IPTG for 30 min, and then rifampicin was added. The incubation was continued further and cells were harvested at the indicated time points for RNA preparation. Total RNA was analyzed using probes specific for uidA mRNA and 16S rRNA respectively. Bacterial cells containing empty vector pRI were used as the control.

 

Figure 8
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  		Figure 8. Effect of RNase E on the functioning of atsRNAs and degradation of target mRNA. (A) The effect of atsRNA CY4 and CY9 on uidA gene in strains CSH26 and its temperature-sensitive rne-1 mutant HAT103. Overnight cultures grown at 30°C were diluted 1:100 into LB medium. Then the bacterial strains were grown at 30°C or 42°C, respectively. The activity of beta-glucuronidase was assayed after the expression of atsRNA was induced with 1 mM IPTG for 30 min. Bacterial cells containing empty vector pRI were used as the control and average values were from triplicate experiments with std values indicated. (B) Quantitative real-time PCR analysis of uidA mRNA in strains grown at 30°C. (C) Quantitative real-time PCR analysis of uidA mRNA in strains grown at 42°C. In (B) and (C), the mRNA level was detected after the expression of atsRNA was induced with 1 mM IPTG for 30 min. Bacterial cells containing empty vector pRI were used as the control. The value was normalized to the level of 16S rRNA in each sample.

 
Characterization of atsRNA targeting against bacterial essential genes
To determine whether atsRNA-mediated gene silencing is suitable for study of bacterial essential gene, a series of atsRNAs targeting against identified essential genes was designed according to the above designing principle. The essential genes murA (encoding UDP-N-acetylglucosamine enolpyruvoyl transferase), trmA (encoding tRNA (m5-U54) methyltransferase) and ygjD (encoding a putative O-sialoglycoprotein endopeptidase) were chosen as the targets (43–45). Twelve atsRNAs were designed and synthesized (M1–M4 for murA, T1–T4 for trmA and Y1–Y4 for ygjD). The sequence and secondary structure of these atsRNAs are listed in Supplementary Data (Supplementary Figure S17 and Table S4). Plasmid pRI carrying atsRNA genes were transformed into E. coli K12 strain MC4100, respectively. The efficacies of these atsRNAs were first determined by QPCR analysis (Supplementary Figure S18). According to the QPCR results, atsRNA M1, T1, Y1 and Y2 reached a reduction over 80% on the expression of target gene and most of the other atsRNAs (except for M4 and Y4) inhibited the expression of target gene obviously (>50%). As the three target genes have been demonstrated to be crucial for bacterial growth rate, we then characterized the effect of atsRNA on cell growth rate by detecting the growth curve of host bacteria. As shown in Figure 9, all the atsRNAs suppressed the growth rate of host bacteria obviously, indicating that atsRNA repressed the expression of target gene efficiently. To further confirm the effect of atsRNAs M1, T1 and Y1, IPTG was added to a final concentration of 1 mM when OD600 reached 0.8 and then the growth curve was detected. As shown in Figure 9D, the bacterial growth rate decreased dramatically after the induction of atsRNA, suggesting that atsRNAs were effective in repressing the expression of target gene. This result demonstrated that the atsRNA-mediated silencing method would be a valuable tool for study of bacterial essential gene in which knockouts are notoriously tedious to perform.


Figure 9
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  		Figure 9. Effect of atsRNAs targeting against essential genes on growth rate of host bacteria. Growth curves of MC4100 strains after the expression of atsRNAs targeting against murA (A), trmA (B) and ygjD (C). Overnight cultures were diluted 1:100 into LB medium and bacterial strains were grown with 1 mM IPTG at time 0 at 30°C. (D) Growth curve of MC4100 strain after the expression of atsRNAs M1, T1 and Y1. Overnight cultures were diluted 1:100 into LB medium and bacterial strains were grown 30°C. When OD600 reached 0.8, 1 mM IPTG was added to induce the expression of atsRNA. Bacterial cells containing empty vector pRI and atsRNA CY2 were used as the control. Turbidity of the cultures was monitored at 600 nm.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
SUPPLEMENTARY DATA
 FUNDING
 REFERENCES
 
In this study, we have developed an effective and convenient method to downregulate the expression of specific gene using atsRNAs in bacteria. atsRNA was designed to be a modular structure composed of mRNA bas-epairing region, Hfq binding site and typical Rho-independent terminator. Using this method, we successfully repressed the expression of exogenous EGFP gene and endogenous uidA gene in E. coli. atsRNA acted by basepairing with the 5' UTR of target mRNA and the atsRNA-mediated gene silencing was Hfq dependent. atsRNA led to translation inhibition and RNase-E-dependent degradation of target mRNA and the translation inhibition was the primary event for gene silencing.

The atsRNA-mediated gene silencing holds many advantages over conventional methods. It is an easy and fast way of downregulating the expression of specific genes, and more importantly, it is an inducible system. The existing methods for functional decipherment of genes in prokaryotes, such as homologous recombination and transposon mutagenesis, significantly limit the study of bacterial essential genes. The atsRNA-mediated silencing is promised to be an alternative RNA tool of deciphering gene function in bacteria when gene knockout was time consuming. In addition, atsRNA acting at post-transcriptional level provides a simple way to impose an overarching level of regulation on a group of genes or operons that may be regulated in many different ways at the level of transcription. Due to its low cost and easy designing, atsRNA makes the automatic and high-throughput functional decipherment of prokaryotic genes possible.

Bacterial sRNAs that act by basepairing can be divided into two classes: cis-encoded and trans-encoded antisense sRNAs. The former are located in the same DNA region and are, therefore, fully complementary to their targets over a long sequence stretch, whereas the latter are located in another chromosomal location, and are only partially complementary to their target mRNAs. The traditional asRNA strategy has been developed based on the cis-encoded asRNAs. Thus, asRNAs were fully complementary to the target genes over a long region (usually over 300 nt). Although the asRNA technology worked well in some cases (46), it usually led to a 40–60% reduction in the expression of target mRNA (47,48). In this study, the atsRNA strategy was developed based on the trans-encoded asRNAs. They were complementary to the target mRNAs over a stretch of ~20 nt (Supplementary Figure S4 and Table S1), and even within this stretch, the complementarity is not complete. The full length of atsRNAs was generally shorter than 100 nt. In this study, the interference efficiency of effective atsRNA could reach over 80%, which is higher than that of asRNAs. Stability was important for the efficacy of both asRNA and atsRNA. The half-life of atsRNA as well as the natural trans-encoded sRNAs was usually longer than 20 min (14,41,49), while the half-life of asRNA was usually shorter than 5 min (48,50). This may partially explain the improved efficacy of atsRNA.

The RNA chaperone protein Hfq is important for the functioning of sRNA; it facilitates the interaction between some of well-characterized sRNA and their targets (41,51). Hfq has been shown to exert a stabilizing effect on endogenous sRNAs by protecting them from endonucleolytic cleavage (52,53). Our results demonstrated that Hfq was also required for the stability of atsRNAs. The interaction between Hfq and atsRNA was presumed to increase the stability and then enhance the efficacy of atsRNA. Hfq appears to bind preferentially to unstructured AU-rich regions, frequently between more structured loop regions of sRNA (14,15,37). In addition, the AU-rich Hfq binding site of atsRNA has been demonstrated to be crucial for the binding affinity to Hfq (Supplementary Figure S14). In this study, atsRNAs with long putative Hfq binding site in secondary structure, such as GY2, GY6, CY4 and CY9, had higher interference efficiency than those with short putative Hfq binding site (e.g. GY3, GY5 and CY6). Therefore, the length of putative Hfq binding site was proposed to be crucial for the interaction between atsRNA and Hfq. In order to enhance the efficacy of atsRNA, the putative Hfq binding site could be appropriately elongated during the designing process. However, longer putative Hfq binding site is not always better. For instance, increase of the length of putative Hfq binding site of CY9 did not result in an increase of interference efficiency. According to our results, a length of 10 nt of putative Hfq binding site was believed to be enough for the functioning of atsRNA.

The efficacy of atsRNA depends on several factors, including the structure and stability itself, the binding kinetics (e.g. binding rate and binding strength) between atsRNA and target mRNA and the Hfq binding affinity. The lower inhibitory efficiency of CY1 and CY10 was presumed to be due to the unstable structure according to the predicted minimum free energy. Since the short putative Hfq binding sites, the less effective efficiency of GY3, GY5, CY3 and CY6 was hypothesized to be due to the low binding affinity of Hfq. The secondary structure of atsRNA is an important factor for inhibitory efficiency of atsRNA. Our results indicated the stem–loop structure within the mRNA base-pairing region is necessary for atsRNA regulation. Especially, the size of the loop structure is crucial for the activity of atsRNA (Figure 3). However, the optimal number of unpaired nucleotides in the loop is not always the same for every atsRNA. For instance, the optimal number of loop nucleotides for atsRNA GY2, GY6 and CY4 was 9 nt, 11 nt and 8 nt, respectively (data not shown). We presume that the optimal size of the loop depends on the secondary structure of both atsRNA and target mRNA, as well as the nucleotide composition in the loop. In addition, according to the calculated thermodynamic pairing energy value of atsRNA–target duplex formation, there is no direct correlation between the inhibitory efficiency and the stability of the hybrids (Supplementary Figure S4). In is worth noting that the inhibitory efficiency of classical cis-acting asRNAs has proven to be primarily controlled by the binding rate constant rather than the binding affinity between the sense–antisense complexes (54). However, the relationship between inhibitory efficiency and binding kinetics of atsRNA requires further studies. Since the participation of Hfq in atsRNA regulation, the binding kinetic features of atsRNA would be more complex than that of cis-acted asRNA. In this study, the number of hybrid base pairings of atsRNA ranged mainly from 19 to 23 nt in length. Some effective asRNA cassettes with a small number of base pairings (12 nt) have been reported, indicating that relatively short antisense sequences can also suffice for efficient binding and regulation (48). However, in the absence of knowledge about target accessibility, we still favor large number of base pairing sequences during atsRNA design process, which are more likely to contain structures that efficiently seed hybridization. Furthermore, a large number of antisense nucleotides would be helpful for the inhibitory specificity of atsRNA.

In this study, the effective atsRNA could result in a 4–7-fold reduction on target genes, and this is enough for functional investigation of bacterial genes. However, overexpression of natural trans-encoded sRNAs could generally repress the target genes from 7–100-fold (11,55–58). Therefore, we will further optimize the above-discussed design parameters to increase the efficacy of atsRNA. More importantly, research progress on natural trans-encoded sRNA will be contributive to the improvement of atsRNA technology. Additionally, taking into account the structural accessibility of target mRNA during the design process would be helpful for the efficacy of atsRNA. Since the binding between many natural sRNAs and their corresponding targets initiates through a loop–loop structure (39,59–61), the target site for loop nucleotides of atsRNA should also be located in an accessible loop structure.

In E. coli, the endogenous sRNAs SgrS and RyhB have been shown clearly to form a specific ribonucleoprotein complex with RNase E and Hfq, resulting in translation inhibition and RNase-E-dependent degradation of target mRNAs (62,63). Translation inhibition is the primary event for gene silencing rather than the target mRNA degradation (64). In this study, atsRNA also led to translational repression and RNase-E-dependent degradation of target mRNA and gene silencing occurred in the absence of target mRNA degradation. This is quite similar with the endogenous sRNAs RyhB and SgrS. According to the mechanism similarity between endogenous trans-encoded sRNA and atsRNA, we conclude that atsRNA functions also by associating with the RNase E/Hfq complex. The atsRNA–Hfq–RNase E complex acts on the SD sequences of target mRNA though base-pairing sequences, resulting in translation inhibition and RNase-E-dependent degradation of target mRNA. For both the two endogenous sRNA and atsRNA, the decay of target mRNAs is just a consequence of translational inhibition. However, the physiological relevance of the RNase-E-dependent degradation of target mRNAs would be necessary to make gene silencing irreversible.

The small RNA has become a standard experimental tool and its therapeutic potential is being aggressively harnessed in eukaryotes. Similarly, the atsRNA-mediated gene silencing will become a popular method for studying gene function and elucidating networks of gene expression in bacteria. Furthermore, recent studies have demonstrated that numerous endogenous trans-encoded sRNAs have crucial roles in bacterial stress responses and virulence regulation (65,66). Therefore, atsRNAs targeting against virulence genes would function effectively in bacterial pathogens and it could potentially serve as antibiotics. Given the emergence and increasing prevalence of bacterial strains that are resistant to available antibiotics, atsRNAs will provide an alternative approach to antimicrobial therapy that offers promising opportunities to inhibit pathogenesis and its consequences without placing immediate life-or-death pressure on the target bacterium.


   SUPPLEMENTARY DATA
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY DATA
FUNDING
 REFERENCES
 
Supplementary Data are available at NAR Online.


   FUNDING
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY DATA
 FUNDING
REFERENCES
 
The National Basic Research Program of China (973 Program; 2003CB716402); and the National High Technology Research and Development Program of China (863 Program; 2007AA09Z418). Funding for open access charge: The National High Technology Research and Development Program of China.

Conflict of interest statement. None declared.


   ACKNOWLEDGEMENTS
 
We would like to thank Gisela Storz (National Institutes of Health), R. Gary Sawers (Martin-Luther University Halle-Wittenberg) and Masaaki Wachi (Tokyo Institute of Technology) for providing plasmid and strains. We thank Guanpin Yang (Ocean University of China) for advice and comments on the manuscript.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY DATA
 FUNDING
 REFERENCES
 

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