An Effective Method for Specific Gene Silencing in Escherichia coli Using Artificial Small RNA.

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.

The small RNA, SdsR, acts as a novel type of toxin in Escherichia coli.

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.

Regulation of BC200 RNA-mediated translation inhibition by hnRNP E1 and E2.

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.

Identification of novel sRNAs involved in biofilm formation, motility, and fimbriae formation in Escherichia coli.

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.

An Effective Method for Specific Gene Silencing in Escherichia coli Using Artificial Small RNA.

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.

Systematic analysis of the role of bacterial Hfq-interacting sRNAs in the response to antibiotics.

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.

SET7/9 methylation of the pluripotency factor LIN28A is a nucleolar localization mechanism that blocks let-7 biogenesis in human ESCs.

LIN28-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.

Electrophoretic mobility shift assay of RNA-RNA complexes.

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.

Roles of rpoS-activating small RNAs in pathways leading to acid resistance of Escherichia coli.

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.

Regulation of transcription from two ssrS promoters in 6S RNA biogenesis.

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.

Exploring sRNA-mediated gene silencing mechanisms using artificial small RNAs derived from a natural RNA scaffold in Escherichia coli.

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.

The CnuK9E H-NS complex antagonizes DNA binding of DicA and leads to temperature-dependent filamentous growth in E. coli.

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°C. This variant (CnuK9E) bears a substitution of a lysine to glutamic acid, causing a charge reversal in the first helix.

Recognition and discrimination of target mRNAs by Sib RNAs, a cis-encoded sRNA family.

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.

On-off controllable RNA hybrid expression vector for yeast three-hybrid system.

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.