TY - JOUR A1 - Gross, Henrik A1 - Hennard, Christine A1 - Masouris, Ilias A1 - Cassel, Christian A1 - Barth, Stephanie A1 - Stober-Grässer, Ute A1 - Mamiani, Alfredo A1 - Moritz, Bodo A1 - Ostareck, Dirk A1 - Ostareck-Lederer, Antje A1 - Neuenkirchen, Nils A1 - Fischer, Utz A1 - Deng, Wen A1 - Leonhardt, Heinrich A1 - Noessner, Elfriede A1 - Kremmer, Elisabeth A1 - Grässer, Friedrich A. T1 - Binding of the Heterogeneous Ribonucleoprotein K (hnRNP K) to the Epstein-Barr Virus Nuclear Antigen 2 (EBNA2) Enhances Viral LMP2A Expression JF - PLoS One N2 - The Epstein-Barr Virus (EBV) -encoded EBNA2 protein, which is essential for the in vitro transformation of B-lymphocytes, interferes with cellular processes by binding to proteins via conserved sequence motifs. Its Arginine-Glycine (RG) repeat element contains either symmetrically or asymmetrically di-methylated arginine residues (SDMA and ADMA, respectively). EBNA2 binds via its SDMA-modified RG-repeat to the survival motor neurons protein (SMN) and via the ADMA-RG-repeat to the NP9 protein of the human endogenous retrovirus K (HERV-K (HML-2) Type 1). The hypothesis of this work was that the methylated RG-repeat mimics an epitope shared with cellular proteins that is used for interaction with target structures. With monoclonal antibodies against the modified RG-repeat, we indeed identified cellular homologues that apparently have the same surface structure as methylated EBNA2. With the SDMA-specific antibodies, we precipitated the Sm protein D3 (SmD3) which, like EBNA2, binds via its SDMA-modified RG-repeat to SMN. With the ADMA-specific antibodies, we precipitated the heterogeneous ribonucleoprotein K (hnRNP K). Specific binding of the ADMA-antibody to hnRNP K was demonstrated using E. coli expressed/ADMA-methylated hnRNP K. In addition, we show that EBNA2 and hnRNP K form a complex in EBV-infected B-cells. Finally, hnRNP K, when co-expressed with EBNA2, strongly enhances viral latent membrane protein 2A (LMP2A) expression by an unknown mechanism as we did not detect a direct association of hnRNP K with DNA-bound EBNA2 in gel shift experiments. Our data support the notion that the methylated surface of EBNA2 mimics the surface structure of cellular proteins to interfere with or co-opt their functional properties. KW - SM proteins KW - protein argentine methyltranserase KW - motor-neuron protein KW - RNA-polymerase-II KW - messenger RNA KW - C-MYC KW - gene expression KW - splicing factor KW - down regulation KW - living cells Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-133707 VL - 7 IS - 8 ER - TY - JOUR A1 - Ramachandran, Vinoy K. A1 - Shearer, Neil A1 - Jacob, Jobin J. A1 - Sharma, Cynthia M. A1 - Thompson, Arthur T1 - The architecture and ppGpp-dependent expression of the primary transcriptome of Salmonella Typhimurium during invasion gene expression JF - BMC Genomics N2 - Background: Invasion of intestinal epithelial cells by Salmonella enterica serovar Typhimurium (S. Typhimurium) requires expression of the extracellular virulence gene expression programme (STEX), activation of which is dependent on the signalling molecule guanosine tetraphosphate (ppGpp). Recently, next-generation transcriptomics (RNA-seq) has revealed the unexpected complexity of bacterial transcriptomes and in this report we use differential RNA sequencing (dRNA-seq) to define the high-resolution transcriptomic architecture of wildtype S. Typhimurium and a ppGpp null strain under growth conditions which model STEX. In doing so we show that ppGpp plays a much wider role in regulating the S. Typhimurium STEX primary transcriptome than previously recognised. Results: Here we report the precise mapping of transcriptional start sites (TSSs) for 78% of the S. Typhimurium open reading frames (ORFs). The TSS mapping enabled a genome-wide promoter analysis resulting in the prediction of 169 alternative sigma factor binding sites, and the prediction of the structure of 625 operons. We also report the discovery of 55 new candidate small RNAs (sRNAs) and 302 candidate antisense RNAs (asRNAs). We discovered 32 ppGpp-dependent alternative TSSs and determined the extent and level of ppGpp-dependent coding and non-coding transcription. We found that 34% and 20% of coding and non-coding RNA transcription respectively was ppGpp-dependent under these growth conditions, adding a further dimension to the role of this remarkable small regulatory molecule in enabling rapid adaptation to the infective environment. Conclusions: The transcriptional architecture of S. Typhimurium and finer definition of the key role ppGpp plays in regulating Salmonella coding and non-coding transcription should promote the understanding of gene regulation in this important food borne pathogen and act as a resource for future research. KW - legionella pneumophila KW - growth rate control KW - escherichia coli K-12 KW - pathogenicity island 2 KW - bacterial signal molecule KW - enterica serovar typhimurium KW - messenger RNA KW - protein synthesis KW - sationary phase KW - environmental regulation Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-130625 VL - 13 IS - 25 ER - TY - JOUR A1 - Fröhlich, Kathrin S. A1 - Papenfort, Kai A1 - Berger, Allison A. A1 - Vogel, Jörg T1 - A conserved RpoS-dependent small RNA controls the synthesis of major porin OmpD JF - Nucleic Acids Research N2 - A remarkable feature of many small non-coding RNAs (sRNAs) of Escherichia coli and Salmonella is their accumulation in the stationary phase of bacterial growth. Several stress response regulators and sigma factors have been reported to direct the transcription of stationary phase-specific sRNAs, but a widely conserved sRNA gene that is controlled by the major stationary phase and stress sigma factor, Sigma(S) (RpoS), has remained elusive. We have studied in Salmonella the conserved SdsR sRNA, previously known as RyeB, one of the most abundant stationary phase-specific sRNAs in E. coli. Alignments of the sdsR promoter region and genetic analysis strongly suggest that this sRNA gene is selectively transcribed by Sigma(S). We show that SdsR down-regulates the synthesis of the major Salmonella porin OmpD by Hfq-dependent base pairing; SdsR thus represents the fourth sRNA to regulate this major outer membrane porin. Similar to the InvR, MicC and RybB sRNAs, SdsR recognizes the ompD mRNA in the coding sequence, suggesting that this mRNA may be primarily targeted downstream of the start codon. The SdsR-binding site in ompD was localized by 3'-RACE, an experimental approach that promises to be of use in predicting other sRNA-target interactions in bacteria. KW - shock sigma factor KW - general stress response KW - down regulation KW - stationary phase KW - salmonella enterica KW - messenger RNA KW - escherichia coli KW - enterica serovar typhimurium KW - outer-membrane proteins KW - small noncoding RNAs Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-134230 VL - 40 IS - 8 ER - TY - JOUR A1 - Lioliou, Efthimia A1 - Sharma, Cynthia M. A1 - Caldelari, Isabelle A1 - Helfer, Anne-Catherine A1 - Fechter, Pierre A1 - Vandenesch, François A1 - Vogel, Jörg A1 - Romby, Pascale T1 - Global Regulatory Functions of the Staphylococcus aureus Endoribonuclease III in Gene Expression JF - PLoS Genetics N2 - RNA turnover plays an important role in both virulence and adaptation to stress in the Gram-positive human pathogen Staphylococcus aureus. However, the molecular players and mechanisms involved in these processes are poorly understood. Here, we explored the functions of S. aureus endoribonuclease III (RNase III), a member of the ubiquitous family of double-strand-specific endoribonucleases. To define genomic transcripts that are bound and processed by RNase III, we performed deep sequencing on cDNA libraries generated from RNAs that were co-immunoprecipitated with wild-type RNase III or two different cleavage-defective mutant variants in vivo. Several newly identified RNase III targets were validated by independent experimental methods. We identified various classes of structured RNAs as RNase III substrates and demonstrated that this enzyme is involved in the maturation of rRNAs and tRNAs, regulates the turnover of mRNAs and non-coding RNAs, and autoregulates its synthesis by cleaving within the coding region of its own mRNA. Moreover, we identified a positive effect of RNase III on protein synthesis based on novel mechanisms. RNase III–mediated cleavage in the 5′ untranslated region (5′UTR) enhanced the stability and translation of cspA mRNA, which encodes the major cold-shock protein. Furthermore, RNase III cleaved overlapping 5′UTRs of divergently transcribed genes to generate leaderless mRNAs, which constitutes a novel way to co-regulate neighboring genes. In agreement with recent findings, low abundance antisense RNAs covering 44% of the annotated genes were captured by co-immunoprecipitation with RNase III mutant proteins. Thus, in addition to gene regulation, RNase III is associated with RNA quality control of pervasive transcription. Overall, this study illustrates the complexity of post-transcriptional regulation mediated by RNase III. KW - staphylococcus aureus KW - ribonucleases KW - messenger RNA KW - RNA sequencing KW - antisense RNA KW - RNA structure KW - RNA synthesis KW - RNA denaturation Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-127219 VL - 8 IS - 6 ER -