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  • 1
    In: EMBO Journal, 17 October 2012, Vol.31(20), pp.4005-4019
    Description: The small RNAs associated with the protein Hfq constitute one of the largest classes of post‐transcriptional regulators known to date. Most previously investigated members of this class are encoded by conserved free‐standing genes. Here, deep sequencing of Hfq‐bound transcripts from multiple stages of growth of revealed a plethora of new small RNA species from within mRNA loci, including DapZ, which overlaps with the 3′ region of the biosynthetic gene, . Synthesis of the DapZ small RNA is independent of DapB protein synthesis, and is controlled by HilD, the master regulator of invasion genes. DapZ carries a short G/U‐rich domain similar to that of the globally acting GcvB small RNA, and uses GcvB‐like seed pairing to repress translation of the major ABC transporters, DppA and OppA. This exemplifies double functional output from an mRNA locus by the production of both a protein and an Hfq‐dependent ‐acting RNA. Our atlas of Hfq targets suggests that the 3′ regions of mRNA genes constitute a rich reservoir that provides the Hfq network with new regulatory small RNAs. Deep sequencing of Hfq‐binding RNAs isolated from at different growth stages reveals that the 3′ UTR of bacterial mRNAs are a rich source of regulatory small RNAs which modulate gene expression in trans.
    Keywords: Abc Transporter ; Dapz ; Gcvb ; Hfq ; 3′ Utr
    ISSN: 0261-4189
    E-ISSN: 1460-2075
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  • 2
    Language: English
    In: RNA Biology, 01 April 2012, Vol.9(4), pp.520-531
    Description: Helicobacter pylori, one of the most prevalent human pathogens, used to be thought to lack small regulatory RNAs (sRNAs) which are otherwise considered abundant in all bacteria. However, our recent analysis of the primary transcriptome of H. pylori discovered an unexpectedly large number of sRNAs, and suggested that this model organism also uses riboregulation to control the expression of its genes. Nonetheless, whereas most enterobacterial sRNAs require the RNA chaperone Hfq for function, Epsilonproteobacteria including H. pylori seem to have no Hfq homologue, which prompted us to search for other auxiliary proteins in sRNA-mediated regulation. Therefore, we have developed two orthogonal methods to isolate and investigate in vivo and in vitro assembled RNA-protein complexes in H. pylori: (i) an affinity chromatography strategy based on aptamer-tagged sRNAs of interest to identify their protein binding partners; and (ii) a rapid method for chromosomal FLAG-tagging of proteins to facilitate co-immunoprecipitation of associated RNA species. Using these methods, we have identified RNA-protein interactions between the ribosomal protein S1 and various mRNAs and sRNAs of H. pylori. Moreover, both methods reported a stable RNA-protein complex between the abundant HPnc6910 sRNA and HP1334, a protein of unknown function that is encoded downstream of HPnc6910. Given that 50% of all bacteria may lack Hfq, our methods can be useful to identify RNA-protein interactions in a wider range of bacterial pathogens.
    Keywords: RNA-Seq ; Small RNA ; Hfq ; Helicobacter Pylori ; RNA Binding Proteins ; Affinity Chromatography ; Post-Transcriptional Control ; Co-Immunoprecipitation ; Anatomy & Physiology
    ISSN: 1547-6286
    E-ISSN: 1555-8584
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  • 3
    Language: English
    In: Methods in molecular biology (Clifton, N.J.), 2012, Vol.905, pp.177-200
    Description: Small regulatory RNAs (sRNAs) are short, generally noncoding RNAs that act posttranscriptionally to control target gene expression. Over the past 10 years there has been a rapid expansion in the discovery and characterization of sRNAs in a diverse range of bacteria. Paradigm shifts in our understanding of the breadth of posttranscriptional control by sRNAs were achieved in a number of pioneering studies that involved immunoprecipitation of a known RNA chaperone, the near-ubiquitous Hfq, followed by sequencing to identify novel putative regulators and targets. To perform the converse experiment, we previously developed a method which uses an aptamer-tagged sRNA to allow purification of in vivo assembled RNA-protein complexes and subsequent identification of bound proteins. We successfully implemented this protocol using the Hfq-associated sRNA, InvR, tagged with a tandem repeat of the commonly used MS2-aptamer. Incorporation of the aptamer had no effect on sRNA stability or activity. InvR-MS2 could be effectively purified along with associated proteins, such as Hfq, using maltose binding protein fused to the MS2 coat protein (MBP-MS2) immobilized on an amylose column. Mass-spectroscopy was also used to identify previously uncharacterized protein partners. These results have been described previously (Said et al., Nucleic Acids Res 37:e133, 2009) and thus the figures presented here are intended solely as an illustrative guide to complement this detailed step-by-step protocol.
    Keywords: Aptamers, Nucleotide -- Metabolism ; RNA, Small Untranslated -- Metabolism ; RNA-Binding Proteins -- Metabolism
    ISBN: 9781617799488
    ISSN: 10643745
    E-ISSN: 1940-6029
    Source: MEDLINE/PubMed (U.S. National Library of Medicine)
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