Gene silencing in Escherichia coli using antisense RNAs expressed from doxycycline‐inducible vectors

Here, we report on the construction of doxycycline (tetracycline analogue)‐inducible vectors that express antisense RNAs in Escherichia coli. Using these vectors, the expression of genes of interest can be silenced conditionally. The expression of antisense RNAs from the vectors was more tightly regulated than the previously constructed isopropyl‐β‐D‐galactopyranoside‐inducible vectors. Furthermore, expression levels of antisense RNAs were enhanced by combining the doxycycline‐inducible promoter with the T7 promoter‐T7 RNA polymerase system; the T7 RNA polymerase gene, under control of the doxycycline‐inducible promoter, was integrated into the lacZ locus of the genome without leaving any antibiotic marker. These vectors are useful for investigating gene functions or altering cell phenotypes for biotechnological and industrial applications.

Significance and Impact of the Study

A gene silencing method using antisense RNAs in Escherichia coli is described, which facilitates the investigation of bacterial gene function. In particular, the method is suitable for comprehensive analyses or phenotypic analyses of genes essential for growth. Here, we describe expansion of vector variations for expressing antisense RNAs, allowing choice of a vector appropriate for the target genes or experimental purpose.     

Conditional gene silencing of multiple genes with antisense RNAs and generation of a mutator strain of Escherichia coli

In this study, we describe a method of simultaneous conditional gene silencing of up to four genes in Escherichia coli by using antisense RNAs. We used antisense RNAs with paired termini, which carried flanking inverted repeats to create paired double-stranded RNA termini; these RNAs have been proven to have high silencing efficacy. To express antisense RNAs, we constructed four IPTG-inducible vectors carrying different but compatible replication origins. When the lacZ antisense RNA was expressed using these vectors, lacZ expression was successfully silenced by all the vectors, but the expression level of the antisense RNA and silencing efficacy differed depending on the used vectors. All the vectors were co-transformable; the antisense RNAs against lacZ, ackA, pta and pepN were co-expressed, and silencing of all the target genes was confirmed. Furthermore, when antisense RNAs were targeted to the mutator genes mutS, mutD (dnaQ) and ndk, which are involved in DNA replication or DNA mismatch repair, spontaneous mutation frequencies increased over 2000-fold. The resulting mutator strain is useful for random mutagenesis of plasmids. The method provides a robust tool for investigating functional relationships between multiple genes or altering cell phenotypes for biotechnological and industrial applications.     

Dual-function RNA regulators in bacteria

The importance of small RNA (sRNA) regulators has been recognized across all domains of life. In bacteria, sRNAs typically control the expression of virulence and stress response genes via antisense base pairing with mRNA targets. Originally dubbed “non-coding RNAs,” a number of bacterial antisense sRNAs have been found to encode functional proteins. Although very few of these dual-function sRNAs have been characterized, they have been found in both gram-negative and gram-positive organisms. Among the few known examples, the functions and mechanisms of regulation by dual-function sRNAs are variable. Some dual-function sRNAs depend on the RNA chaperone Hfq for base pairing-dependent regulation (riboregulation); this feature appears so far exclusive to gram-negative bacterial sRNAs. Other variations can be found in the spatial organization of the coding region with respect to the riboregulation determinants. How the functions of encoded proteins relate to riboregulation is for the most part not understood. However, in one case it appears that there is physiological redundancy between protein and riboregulation functions. This mini-review focuses on the two best-studied bacterial dual-function sRNAs: RNAIII from Staphylococcus aureus and SgrS from Escherichia coli and includes a discussion of what is known about the structure, function and physiological roles of these sRNAs as well as what questions remain outstanding.     

Programmable control of bacterial gene expression with the combined CRISPR and antisense RNA system

A central goal of synthetic biology is to implement diverse cellular functions by predictably controlling gene expression. Though research has focused more on protein regulators than RNA regulators, recent advances in our understanding of RNA folding and functions have motivated the use of RNA regulators. RNA regulators provide an advantage because they are easier to design and engineer than protein regulators, potentially have a lower burden on the cell and are highly orthogonal. Here, we combine the CRISPR system from Streptococcus pyogenes and synthetic antisense RNAs (asRNAs) in Escherichia coli strains to repress or derepress a target gene in a programmable manner. Specifically, we demonstrate for the first time that the gene target repressed by the CRISPR system can be derepressed by expressing an asRNA that sequesters a small guide RNA (sgRNA). Furthermore, we demonstrate that tunable levels of derepression can be achieved (up to 95%) by designing asRNAs that target different regions of a sgRNA and by altering the hybridization free energy of the sgRNA–asRNA complex. This new system, which we call the combined CRISPR and asRNA system, can be used to reversibly repress or derepress multiple target genes simultaneously, allowing for rational reprogramming of cellular functions.     

Antisense RNA based down-regulation of RNaseE in E.coli

Background  

Messenger RNA decay is an important mechanism for controlling gene expression in all organisms. The rate of the mRNA degradation directly affects the steady state concentration of mRNAs and therefore influences the protein synthesis. RNaseE has a key importance for the general mRNA decay in E.coli. While RNaseE initiates the degradation of most mRNAs in E.coli, it is likely that the enzyme is also responsible for the degradation of recombinant RNAs. As RNaseE is essential for cell viability and knockout mutants cannot be cultured, we investigated the possibility for a down-regulation of the intracellular level of RNaseE by antisense RNAs. During this study, an antisense RNA based approach could be established which revealed a strong reduction of the intracellular level of RNaseE in E.coli.     

Evidence for a major role of antisense RNAs in cyanobacterial gene regulation

Information on the numbers and functions of naturally occurring antisense RNAs (asRNAs) in eubacteria has thus far remained incomplete. Here, we screened the model cyanobacterium Synechocystis sp. PCC 6803 for asRNAs using four different methods. In the final data set, the number of known noncoding RNAs rose from 6 earlier identified to 60 and of asRNAs from 1 to 73 (28 were verified using at least three methods). Among these, there are many asRNAs to housekeeping, regulatory or metabolic genes, as well as to genes encoding electron transport proteins. Transferring cultures to high light, carbon‐limited conditions or darkness influenced the expression levels of several asRNAs, suggesting their functional relevance. Examples include the asRNA to rpl1, which accumulates in a light‐dependent manner and may be required for processing the L11 r‐operon and the SyR7 noncoding RNA, which is antisense to the murF 5′ UTR, possibly modulating murein biosynthesis. Extrapolated to the whole genome, ～10% of all genes in Synechocystis are influenced by asRNAs. Thus, chromosomally encoded asRNAs may have an important function in eubacterial regulatory networks.     

Identification of novel non-coding RNAs as potential antisense regulators in the archaeon Sulfolobus solfataricus

By generating a specialized cDNA library from the archaeon Sulfolobus solfataricus, we have identified 57 novel small non-coding RNA (ncRNA) candidates and confirmed their expression by Northern blot analysis. The majority was found to belong to one of two classes, either antisense or antisense-box RNAs, where the latter only exhibit partial complementarity to RNA targets. The most prominent group of antisense RNAs is transcribed in the opposite orientation to the transposase genes, encoded by insertion elements (transposons). Thus, these antisense RNAs may regulate transposition of insertion elements by inhibiting expression of the transposase mRNA. Surprisingly, the class of antisense RNAs also contained RNAs complementary to tRNAs or sRNAs (small-nucleolar-like RNAs). For the antisense-box ncRNAs, the majority could be assigned to the class of C/D sRNAs, which specify 2'-O-methylation sites on rRNAs or tRNAs. Five C/D sRNAs of this group are predicted to target methylation at six sites in 13 different tRNAs, thus pointing to the widespread role of these sRNA species in tRNA modification in Archaea. Another group of antisense-box RNAs, lacking typical C/D sRNA motifs, was predicted to target the 3'-untranslated regions of certain mRNAs. Furthermore, one of the ncRNAs that does not show antisense elements is transcribed from a repeat unit of a cluster of small regularly spaced repeats in S. solfataricus which is potentially involved in replicon partitioning. In conclusion, this is the first report of stably expressed antisense RNAs in an archaeal species and it raises the prospect that antisense-based mechanisms are also used widely in Archaea to regulate gene expression.     

Antisense RNA Targeting of Primase Interferes with Bacteriophage Replication in Streptococcus thermophilus

The putative primase gene and other genes associated with the Sfi21-prototype genome replication module are highly conserved in Streptococcus thermophilus bacteriophages. Expression of antisense RNAs complementary to the putative primase gene (pri3.1) from S. thermophilus phage κ3 provided significant protection from κ3 and two other Sfi21-type phages. Expression of pri3.10-AS, an antisense RNA that covered the entire primase gene, reduced the efficiency of plaquing (EOP) of κ3 to 3 × 10⁻³ and reduced its burst size by 20%. Mutant phages capable of overcoming antisense inhibition were not recovered. Thirteen primase-specific antisense cassettes of different lengths (478 to 1,512 bp) were systematically designed to target various regions of the gene. Each cassette conferred some effect, reducing the EOP to between 0.8 and 3 × 10⁻³. The largest antisense RNAs (1.5 kb) were generally found to confer the greatest reductions in EOP, but shorter (0.5 kb) antisense RNAs were also effective, especially when directed to the 5′ region of the gene. The impacts of primase-targeted antisense RNAs on phage development were examined. The expression of pri3.10-AS resulted in reductions in target RNA abundance and the number of phage genomes synthesized. Targeting a key genome replication function with antisense RNA provided effective phage protection in S. thermophilus.     

Effects of different target sites on antisense RNA-mediated regulation of gene expression

Antisense RNA is a type of noncoding RNA (ncRNA) that binds to complementary mRNA sequences and induces gene repression by inhibiting translation or degrading mRNA. Recently, several small ncRNAs (sRNAs) have been identified in Escherichia coli that act as antisense RNA mainly via base pairing with mRNA. The base pairing predominantly leads to gene repression, and in some cases, gene activation. In the current study, we examined how the location of target sites affects sRNA-mediated gene regulation. An efficient antisense RNA expression system was developed, and the effects of antisense RNAs on various target sites in a model mRNA were examined. The target sites of antisense RNAs suppressing gene expression were identified, not only in the translation initiation region (TIR) of mRNA, but also at the junction between the coding region and 3'' untranslated region. Surprisingly, an antisense RNA recognizing the upstream region of TIR enhanced gene expression through increasing mRNA stability. [BMB Reports 2014; 47(11): 619-624]     

The small RNA content of human sperm reveals pseudogene-derived piRNAs complementary to protein-coding genes

At the end of mammalian sperm development, sperm cells expel most of their cytoplasm and dispose of the majority of their RNA. Yet, hundreds of RNA molecules remain in mature sperm. The biological significance of the vast majority of these molecules is unclear. To better understand the processes that generate sperm small RNAs and what roles they may have, we sequenced and characterized the small RNA content of sperm samples from two human fertile individuals. We detected 182 microRNAs, some of which are highly abundant. The most abundant microRNA in sperm is miR-1246 with predicted targets among sperm-specific genes. The most abundant class of small noncoding RNAs in sperm are PIWI-interacting RNAs (piRNAs). Surprisingly, we found that human sperm cells contain piRNAs processed from pseudogenes. Clusters of piRNAs from human testes contain pseudogenes transcribed in the antisense strand and processed into small RNAs. Several human protein-coding genes contain antisense predicted targets of pseudogene-derived piRNAs in the male germline and these piRNAs are still found in mature sperm. Our study provides the most extensive data set and annotation of human sperm small RNAs to date and is a resource for further functional studies on the roles of sperm small RNAs. In addition, we propose that some of the pseudogene-derived human piRNAs may regulate expression of their parent gene in the male germline.     

A Vector Library for Silencing Central Carbon Metabolism Genes with Antisense RNAs in Escherichia coli

We describe here the construction of a series of 71 vectors to silence central carbon metabolism genes in Escherichia coli. The vectors inducibly express antisense RNAs called paired-terminus antisense RNAs, which have a higher silencing efficacy than ordinary antisense RNAs. By measuring mRNA amounts, measuring activities of target proteins, or observing specific phenotypes, it was confirmed that all the vectors were able to silence the expression of target genes efficiently. Using this vector set, each of the central carbon metabolism genes was silenced individually, and the accumulation of metabolites was investigated. We were able to obtain accurate information on ways to increase the production of pyruvate, an industrially valuable compound, from the silencing results. Furthermore, the experimental results of pyruvate accumulation were compared to in silico predictions, and both sets of results were consistent. Compared to the gene disruption approach, the silencing approach has an advantage in that any E. coli strain can be used and multiple gene silencing is easily possible in any combination.     

Small Toxic Proteins and the Antisense RNAs That Repress Them

Summary: There has been a great expansion in the number of small regulatory RNAs identified in bacteria. Some of these small RNAs repress the synthesis of potentially toxic proteins. Generally the toxin proteins are hydrophobic and less than 60 amino acids in length, and the corresponding antitoxin small RNA genes are antisense to the toxin genes or share long stretches of complementarity with the target mRNAs. Given their short length, only a limited number of these type I toxin-antitoxin loci have been identified, but it is predicted that many remain to be found. Already their characterization has given insights into regulation by small RNAs, has suggested functions for the small toxic proteins at the cell membrane, and has led to practical applications for some of the type I toxin-antitoxin loci.     

Reproducible features of small RNAs in C. elegans reveal NU RNAs and provide insights into 22G RNAs and 26G RNAs

Small RNAs regulate gene expression and most genes in the worm Caenorhabditis elegans are subject to their regulation. Here, we analyze small RNA data sets and use reproducible features of RNAs present in multiple data sets to discover a new class of small RNAs and to reveal insights into two known classes of small RNAs—22G RNAs and 26G RNAs. We found that reproducibly detected 22-nt RNAs, although are predominantly RNAs with a G at the 5′ end, also include RNAs with A, C, or U at the 5′ end. These RNAs are synthesized downstream from characteristic sequence motifs on mRNA and have U-tailed derivatives. Analysis of 26G RNAs revealed that they are processed from a blunt end of double-stranded RNAs and that production of one 26G RNA generates a hotspot immediately downstream for production of another. To our surprise, analysis of RNAs shorter than 18 nt revealed a new class of RNAs, which we call NU RNAs (pronounced “new RNAs”) because they have a NU bias at the 5′ end, where N is any nucleotide. NU RNAs are antisense to genes and originate downstream from U bases on mRNA. Although many genes have complementary NU RNAs, their genome-wide distribution is distinct from that of previously known classes of small RNAs. Our results suggest that current approaches underestimate reproducibly detected RNAs that are shorter than 18 nt, and theoretical considerations suggest that such shorter RNAs could be used for sequence-specific gene regulation in organisms like C. elegans that have small genomes.     

Unveiling the biotransformation mechanism of indole in a Cupriavidus sp. strain

Small RNAs (sRNAs), particularly those that act by limited base pairing with mRNAs, are part of most regulatory networks in bacteria. In many cases, the base‐pairing interaction is facilitated by the RNA chaperone Hfq. However, not all bacteria encode Hfq and some base‐pairing sRNAs do not require Hfq raising the possibility of other RNA chaperones. Candidates are proteins with homology to FinO, a factor that promotes base pairing between the FinP antisense sRNA and the traJ mRNA to control F plasmid transfer. Recent papers have shown that the Salmonella enterica FinO‐domain protein ProQ binds a large suite of sRNAs, including the RaiZ sRNA, which represses translation of the hupA mRNA, and the Legionella pneumophila protein RocC binds the RocR sRNA, which blocks expression of competence genes. Here we discuss what is known about FinO‐domain structures, including the recently solved Escherichia coli ProQ structure, as well as the RNA binding properties of this family of proteins and evidence they act as chaperones. We compare these properties with those of Hfq. We further summarize what is known about the physiological roles of FinO‐domain proteins and enumerate outstanding questions whose answers will establish whether they constitute a second major class of RNA chaperones.