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  • 1
    Language: English
    In: Nucleic acids research, 2007, Vol.35(3), pp.1018-37
    Description: Small non-coding RNAs (sRNAs) are an emerging class of regulators of bacterial gene expression. Most of the regulatory Escherichia coli sRNAs known to date modulate translation of trans-encoded target mRNAs. We studied the specificity of sRNA target interactions using gene fusions to green fluorescent protein (GFP) as a novel reporter of translational control by bacterial sRNAs in vivo. Target sequences were selected from both monocistronic and polycistronic mRNAs. Upon expression of the cognate sRNA (DsrA, GcvB, MicA, MicC, MicF, RprA, RyhB, SgrS and Spot42), we observed highly specific translation repression/activation of target fusions under various growth conditions. Target regulation was also tested in mutants that lacked Hfq or RNase III, or which expressed a truncated RNase E (rne701). We found that translational regulation by these sRNAs was largely independent of full-length RNase E, e.g. despite the fact that ompA fusion mRNA decay could no longer be promoted by MicA. This is the first study in which multiple well-defined E.coli sRNA target pairs have been studied in a uniform manner in vivo. We expect our GFP fusion approach to be applicable to sRNA targets of other bacteria, and also demonstrate that Vibrio RyhB sRNA represses a Vibrio sodB fusion when co-expressed in E.coli.
    Keywords: Gene Expression Regulation, Bacterial ; Protein Biosynthesis ; Escherichia Coli -- Genetics ; RNA, Untranslated -- Metabolism
    ISSN: 03051048
    E-ISSN: 1362-4962
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  • 2
    In: Molecular Microbiology, May 2012, Vol.84(3), pp.428-445
    Description: MicF is a textbook example of a small regulatory RNA (sRNA) that acts on a ‐encoded target mRNA through imperfect base pairing. Discovery of MicF as a post‐transcriptional repressor of the major porin OmpF established the paradigm for a meanwhile common mechanism of translational inhibition, through antisense sequestration of a ribosome binding site. However, whether MicF regulates additional genes has remained unknown for almost three decades. Here, we have harnessed the new superfolder variant of GFP for reporter–gene fusions to validate newly predicted targets of MicF in . We show that the conserved 5′ end of MicF acts by seed pairing to repress the mRNAs of global transcriptional regulator Lrp, and periplasmic protein YahO, while a second targeting region is also required to regulate the mRNA of the lipid A‐modifying enzyme LpxR. Interestingly, MicF targets at both the ribosome binding site and deep within the coding sequence. MicF binding in the coding sequence of decreases mRNA stability through exacerbating the use of a native RNase E site proximal to the short MicF‐ duplex. Altogether, this study assigns the classic MicF sRNA to the growing class of Hfq‐associated regulators that use diverse mechanisms to impact multiple loci.
    Keywords: Gene Expression Regulation, Bacterial ; Bacterial Proteins -- Genetics ; Green Fluorescent Proteins -- Metabolism ; Porins -- Genetics ; RNA, Bacterial -- Metabolism ; RNA, Small Untranslated -- Metabolism ; Salmonella Typhimurium -- Metabolism;
    ISSN: 0950-382X
    E-ISSN: 1365-2958
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  • 3
    Language: English
    In: ACS chemical biology, 21 January 2011, Vol.6(1), pp.61-74
    Description: The display and analysis of proteins expressed on biological surfaces has become an attractive tool for the study of molecular interactions in enzymology, protein engineering, and high-throughput screening. Among the growing number of established display systems, retroviruses offer a unique and fully mammalian platform for the expression of correctly folded and post-translationally modified proteins in the context of cell plasma membrane-derived particles. This is of special interest for therapeutic applications such as gene therapy and vaccine development and also offers advantages for the engineering of mammalian proteins toward customized binding affinities and catalytic activities. This review critically summarizes the basic concepts and applications of retroviral display and analyses its benefits in comparison to other display techniques.
    Keywords: Genetic Therapy -- Methods ; Genetic Vectors -- Metabolism ; Protein Engineering -- Methods ; Retroviridae -- Genetics ; Vaccines -- Genetics
    ISSN: 15548929
    E-ISSN: 1554-8937
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  • 4
    Language: English
    In: Nucleic acid therapeutics, June 2016, Vol.26(3), pp.173-82
    Description: Although the use of RNAs has enormous therapeutic potential, these RNA-based therapies can trigger unwanted inflammatory responses by the activation of pattern recognition receptors (PRRs) and cause harmful side effects. In contrast, the immune activation by therapeutic RNAs can be advantageous for treating cancers. Thus, the immunogenicity of therapeutic RNAs should be deliberately controlled depending on the therapeutic applications of RNAs. In this study, we demonstrated that RNAs containing 2'fluoro (2'F) pyrimidines differentially controlled the activation of PRRs. The activity of RNAs that stimulate toll-like receptors 3 and 7 was abrogated by the incorporation of 2'F pyrimidine. By contrast, incorporation of 2'F pyrimidines enhanced the activity of retinoic acid-inducible gene 1-stimulating RNAs. Furthermore, we found that transfection with RNAs containing 2'F pyrimidine and 5' triphosphate (5'ppp) increased cell death and interferon-β expression in human cancer cells compared with transfection with 2'hydroxyl 5'ppp RNAs, whereas RNAs containing 2'O-methyl pyrimidine and 5'ppp completely abolished the induction of cell death and cytokine expression in the cells. Our findings suggest that incorporation of 2'F and 2'O-methyl nucleosides is a facile approach to differentially control the ability of therapeutic RNAs to activate or limit immune and inflammatory responses depending on therapeutic applications.
    Keywords: Floxuridine -- Pharmacology ; RNA -- Chemistry ; Receptors, Pattern Recognition -- Drug Effects
    ISSN: 21593337
    E-ISSN: 2159-3345
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  • 5
    Language: English
    In: Journal of Molecular Biology, 26 October 2007, Vol.373(3), pp.521-528
    Description: Many bacterial genes of related function are organized in operons and transcribed as polycistronic mRNAs to ensure the coordinate expression of the individual cistrons. Post-transcriptional modulation of such mRNAs can alter the expression of downstream cistrons, resulting in discoordinate protein synthesis from an operon mRNA. Several factors, including small non-coding RNAs (sRNAs), have been described that act collectively as repressors within polycistronic mRNAs. We describe the first case of discoordinated operon expression in which a downstream cistron is activated at the post-transcriptional level. We report that GlmY sRNA activates GlmS synthesis from the mRNA without altering GlmU expression. The sRNA is shown to be structurally and functionally conserved in diverse enterobacteria; its transcription may be controlled by the alternative sigma factor, σ . Our data suggest that Gram-negative bacteria evolved a mechanism of riboregulation that is distinct from the riboswitch mechanism of Gram-positive bacteria.
    Keywords: Small Non-Coding RNA ; Glms ; Discoordinate Operon Expression ; Sigma 54 ; Biology ; Chemistry
    ISSN: 0022-2836
    E-ISSN: 1089-8638
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  • 6
    Language: English
    In: PLoS Biology, 2008, Vol.6(3), p.e64
    Description: Small noncoding RNAs (sRNA) can function as posttranscriptional activators of gene expression to regulate stress responses and metabolism. We here describe the mechanisms by which two sRNAs, GlmY and GlmZ, activate the Escherichia coli glmS mRNA, coding for an essential enzyme in amino-sugar metabolism. The two sRNAs, although being highly similar in sequence and structure, act in a hierarchical manner. GlmZ, together with the RNA chaperone, Hfq, directly activates glmS mRNA translation by an anti-antisense mechanism. In contrast, GlmY acts upstream of GlmZ and positively regulates glmS by antagonizing GlmZ RNA inactivation. We also report the first example, to our knowledge, of mRNA expression being controlled by the poly(A) status of a chromosomally encoded sRNA. We show that in wild-type cells, GlmY RNA is unstable due to 3′ end polyadenylation; whereas in an E. coli pcnB mutant defective in RNA polyadenylation, GlmY is stabilized and accumulates, which in turn stabilizes GlmZ and causes GlmS overproduction. Our study reveals hierarchical action of two well-conserved sRNAs in a complex regulatory cascade that controls the glmS mRNA. Similar cascades of noncoding RNA regulators may operate in other organisms. ; Hierarchical action of regulators is a fundamental principle in gene expression control, and is well understood in protein-based signaling pathways. We have discovered that small noncoding RNAs (sRNAs), a new class of gene expression regulators, can also act hierarchically and form a regulatory cascade. Two highly similar sRNAs function after transcription to activate the mRNA, which codes for an essential function in amino-sugar metabolism. It is somewhat unusual for two sRNAs to act upon the same target mRNA, and despite their seeming homology, these two sRNAs (GlmY and GlmZ) employ different molecular mechanisms and function hierarchically to activate expression: GlmZ directly activates translation via disruption of an mRNA structure that inhibits translation, whereas GlmY controls the processing of GlmZ to prevent the inactivation of this direct activator. We also found that GlmY is itself controlled by an RNA processing event (3′ end polyadenylation), which typically destabilizes bacterial RNA. Our data unequivocally demonstrate that is exceptionally dependent on RNA-based mechanisms for its genetic control. Given the large number of noncoding RNAs of unknown function, we believe that similar regulatory RNA cascades may operate in other organisms. ; A regulatory RNA cascade that posttranscriptionally activates the mRNA is identified, with two highly similar small noncoding RNAs acting hierarchically in a manner thus far known only in protein-based regulatory circuits.
    Keywords: Research Article ; Biochemistry ; Genetics And Genomics ; Molecular Biology
    ISSN: 1544-9173
    E-ISSN: 1545-7885
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  • 7
    In: Nature Chemical Biology, 2009, Vol.6(1), p.22
    Description: In an effort to target the in vivo context of tumor-specific moieties, a large library of nuclease-resistant RNA oligonucleotides was screened in tumor-bearing mice to identify candidate molecules with the ability to localize to hepatic colon cancer metastases. One of the selected molecules is an RNA aptamer that binds to protein p68, an RNA helicase that has been shown to be upregulated in colorectal cancer.
    Keywords: Article ; Selection ; Rna Motifs ; Aptamer ; Tumor ; P68 Rna Helicase;
    ISSN: 1552-4450
    E-ISSN: 15524469
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  • 8
    Language: English
    In: Nature Communications, 01 November 2017, Vol.8(1), pp.1-10
    Description: Lanthipeptides are a class of cyclic post-translationally modified peptides with potential drug-like properties. Here the authors develop a phage display system by expressing lanthipeptide precursors as C-terminal fusions to the phage M13 coat protein pIII in E. coli along with the heterologous modifying enzymes.
    Keywords: Biology
    E-ISSN: 2041-1723
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  • 9
    Language: English
    In: Nucleic acids research, 24 February 2005, Vol.33(4), pp.e35
    Description: Antibody library technology represents a powerful tool for the discovery and design of antibodies with high affinity and specificity for their targets. To extend the technique to the expression and selection of antibody libraries in an eukaryotic environment, we provide here a proof of concept that retroviruses can be engineered for the display and selection of variable single-chain fragment (scFv) libraries. A retroviral library displaying the repertoire obtained after a single round of selection of a human synthetic scFv phage display library on laminin was generated. For selection, antigen-bound virus was efficiently recovered by an overlay with cells permissive for infection. This approach allowed more than 10(3)-fold enrichment of antigen binders in a single selection cycle. After three selection cycles, several scFvs were recovered showing similar laminin-binding activities but improved expression levels in mammalian cells as compared with a laminin-specific scFv selected by the conventional phage display approach. Thus, translational problems that occur when phage-selected antibodies have to be transferred onto mammalian expression systems to exert their therapeutic potential can be avoided by the use of retroviral display libraries.
    Keywords: Peptide Library ; Immunoglobulin Variable Region -- Genetics ; Moloney Murine Leukemia Virus -- Genetics
    E-ISSN: 1362-4962
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  • 10
    Language: English
    In: Methods in molecular biology (Clifton, N.J.), 2009, Vol.540, pp.301-19
    Description: Small non-coding RNAs (sRNAs) are an emerging class of regulators of bacterial gene expression, which mainly modulate the translation of trans-encoded mRNAs. Typically, these molecules are 50-200 nucleotides in size and do not contain expressed open reading frames (ORFs). In Escherichia coli, about 70 members of this group have been identified to date and further estimates assume hundreds of sRNAs per bacterial genome. Regulation of gene expression by sRNAs is predominantly mediated by physical sRNA/target mRNA interactions that are based on short and imperfect complementarity. Although the contribution of sRNAs to overall bacterial gene regulation is now being appreciated, the function of many sRNAs is still unknown and their targets await to be uncovered. We recently developed a modular two-plasmid system, based on the green fluorescent protein (GFP) as non-invasive reporter of gene expression, to rapidly monitor the regulatory potential of sRNA/target mRNA pairs under investigation in vivo. The specialized reporter plasmid series also provides a suitable platform to study the function of cis-encoded riboregulators such as natural riboswitches, thermosensors, or engineered aptamer-based regulatory switches.
    Keywords: Gene Expression Regulation, Bacterial ; Transcription, Genetic ; Escherichia Coli -- Genetics ; Green Fluorescent Proteins -- Metabolism ; Molecular Biology -- Methods ; Plasmids -- Genetics
    ISSN: 1064-3745
    Source: MEDLINE/PubMed (U.S. National Library of Medicine)
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