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  • Transcription (Genetics)
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  • 1
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 22 December 2009, Vol.106(51), pp.21878-82
    Description: Methanosarcina mazei and related mesophilic archaea are the only organisms fermenting acetate, methylamines, and methanol to methane and carbon dioxide, contributing significantly to greenhouse gas production. The biochemistry of these metabolic processes is well studied, and genome sequences are available, yet little is known about the overall transcriptional organization and the noncoding regions representing 25% of the 4.01-Mb genome of M. mazei. We present a genome-wide analysis of transcription start sites (TSS) in M. mazei grown under different nitrogen availabilities. Pyrosequencing-based differential analysis of primary vs. processed 5' ends of transcripts discovered 876 TSS across the M. mazei genome. Unlike in other archaea, in which leaderless mRNAs are prevalent, the majority of the detected mRNAs in M. mazei carry long untranslated 5' regions. Our experimental data predict a total of 208 small RNA (sRNA) candidates, mostly from intergenic regions but also antisense to 5' and 3' regions of mRNAs. In addition, 40 new small mRNAs with ORFs of 〈 or = 30 aa were identified, some of which might have dual functions as mRNA and regulatory sRNA. We confirmed differential expression of several sRNA genes in response to nitrogen availability. Inspection of their promoter regions revealed a unique conserved sequence motif associated with nitrogen-responsive regulation, which might serve as a regulator binding site upstream of the common IIB recognition element. Strikingly, several sRNAs antisense to mRNAs encoding transposases indicate nitrogen-dependent transposition events. This global TSS map in archaea will facilitate a better understanding of transcriptional and posttranscriptional control in the third domain of life.
    Keywords: Genes, Archaeal ; Methanosarcina -- Genetics ; Nitrogen -- Metabolism ; RNA, Messenger -- Genetics
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 2
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 2011, Vol.108(5), pp.2124-2129
    Description: There has been an increasing interest in cyanobacteria because these photosynthetic organisms convert solar energy into biomass and because of their potential for the production of biofuels. However, the exploitation of cyanobacteria for bioengineering requires knowledge of their transcriptional organization. Using differential RNA sequencing, we have established a genome-wide map of 3,527 transcriptional start sites (TSS) of the model organism Synechocystis sp. PCC6803. One-third of all TSS were located upstream of an annotated gene; another third were on the reverse complementary strand of 866 genes, suggesting massive antisense transcription. Orphan TSS located in intergenic regions led us to predict 314 noncoding RNAs (ncRNAs). Complementary microarray-based RNA profiling verified a high number of noncoding transcripts and identified strong ncRNA regulations. Thus, ~64% of all TSS give rise to antisense or ncRNAs in a genome that is to 87% protein coding. Our data enhance the information on promoters by a factor of 40, suggest the existence of additional small peptide-encoding mRNAs, and provide corrected 5' annotations for many genes of this cyanobacterium. The global TSS map will facilitate the use of Synechocystis sp. PCC6803 as a model organism for further research on photosynthesis and energy research. ; Includes references ; p. 2124-2129.
    Keywords: Transcription (Genetics) -- Physiological Aspects ; Cyanobacteria -- Genetic Aspects ; Genetic Regulation -- Research ; Rna Polymerases -- Properties;
    ISSN: 0027-8424
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  • 3
    Language: English
    In: Nature, March 11, 2010, Vol.463(7286), p.250(6)
    Description: Genome sequencing of Helicobacter pylori has revealed the potential proteins and genetic diversity of this prevalent human pathogen, yet little is known about its transcriptional organization and noncoding RNA output. Massively parallel cDNA sequencing (RNA-seq) has been revolutionizing global transcriptomic analysis. Here, using a novel differential approach (dRNA-seq) selective for the 5' end of primary transcripts, we present a genome-wide map of H. pylori transcriptional start sites and operons. We discovered hundreds of transcriptional start sites within operons, and opposite to annotated genes, indicating that complexity of gene expression from the small H. pylori genome is increased by uncoupling of polycistrons and by genome-wide antisense transcription. We also discovered an unexpected number of ~60 small RNAs including the e-subdivision counterpart of the regulatory 6S RNA and associated RNA products, and potential regulators of cis- and trans-encoded target messenger RNAs. Our approach establishes a paradigm for mapping and annotating the primary transcriptomes of many living species.
    Keywords: Antisense Rna -- Analysis ; Gene Expression -- Analysis ; Transcription (Genetics) -- Analysis ; Helicobacter Pylori -- Genetic Aspects ; Helicobacter Pylori -- Physiological Aspects
    ISSN: 0028-0836
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  • 4
    Language: English
    In: Journal of bacteriology, November 2012, Vol.194(21), pp.5864-74
    Description: Hfq is an RNA-binding protein known to regulate a variety of cellular processes by interacting with small RNAs (sRNAs) and mRNAs in prokaryotes. Stenotrophomonas maltophilia is an important opportunistic pathogen affecting primarily hospitalized and immunocompromised hosts. We constructed an hfq deletion mutant (Δhfq) of S. maltophilia and compared the behaviors of wild-type and Δhfq S. maltophilia cells in a variety of assays. This revealed that S. maltophilia Hfq plays a role in biofilm formation and cell motility, as well as susceptibility to antimicrobial agents. Moreover, Hfq is crucial for adhesion to bronchial epithelial cells and is required for the replication of S. maltophilia in macrophages. Differential RNA sequencing analysis (dRNA-seq) of RNA isolated from S. maltophilia wild-type and Δhfq strains showed that Hfq regulates the expression of genes encoding flagellar and fimbrial components, transmembrane proteins, and enzymes involved in different metabolic pathways. Moreover, we analyzed the expression of several sRNAs identified by dRNA-seq in wild-type and Δhfq S. maltophilia cells grown in different conditions on Northern blots. The accumulation of two sRNAs was strongly reduced in the absence of Hfq. Furthermore, based on our dRNA-seq analysis we provide a genome-wide map of transcriptional start sites in S. maltophilia.
    Keywords: Host Factor 1 Protein -- Metabolism ; Molecular Chaperones -- Metabolism ; RNA, Bacterial -- Metabolism ; Stenotrophomonas Maltophilia -- Physiology
    ISSN: 00219193
    E-ISSN: 1098-5530
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  • 5
    Language: English
    In: Journal of bacteriology, 01 January 2015, Vol.197(1), pp.18-28
    Description: While the model organism Escherichia coli has been the subject of intense study for decades, the full complement of its RNAs is only now being examined. Here we describe a survey of the E. coli transcriptome carried out using a differential RNA sequencing (dRNA-seq) approach, which can distinguish between primary and processed transcripts, and an automated prediction algorithm for transcriptional start sites (TSS). With the criterion of expression under at least one of three growth conditions examined, we predicted 14,868 TSS candidates, including 5,574 internal to annotated genes (iTSS) and 5,495 TSS corresponding to potential antisense RNAs (asRNAs). We examined expression of 14 candidate asRNAs by Northern analysis using RNA from wild-type E. coli and from strains defective for RNases III and E, two RNases reported to be involved in asRNA processing. Interestingly, nine asRNAs detected as distinct bands by Northern analysis were differentially affected by the rnc and rne mutations. We also compared our asRNA candidates with previously published asRNA annotations from RNA-seq data and discuss the challenges associated with these cross-comparisons. Our global transcriptional start site map represents a valuable resource for identification of transcription start sites, promoters, and novel transcripts in E. coli and is easily accessible, together with the cDNA coverage plots, in an online genome browser.
    Keywords: Escherichia Coli -- Metabolism ; Gene Expression Regulation, Bacterial -- Physiology ; RNA, Antisense -- Metabolism ; RNA, Bacterial -- Metabolism ; Transcription Initiation Site -- Physiology
    ISSN: 00219193
    E-ISSN: 1098-5530
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  • 6
    Language: English
    In: Methods, Sept 15, 2015, Vol.86, p.89(13)
    Description: To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ymeth.2015.06.012 Byline: Thorsten Bischler, Hock Siew Tan, Kay Nieselt, Cynthia M. Sharma Abstract: * A differential RNA-seq (dRNA-seq) approach for primary transcriptome analysis. * dRNA-seq analysis of the gastric pathogen Helicobacter pylori as a model bacterium. * dRNA-seq for global annotation of transcriptional start sites (TSS) and small RNAs. * Comparison of automated and manual TSS annotation. * Variations due to sample and library preparations and sequencing protocols. Article History: Received 8 April 2015; Revised 7 June 2015; Accepted 9 June 2015
    Keywords: Transcription (Genetics) – Genetic Aspects ; Transcription (Genetics) – Analysis ; Helicobacter Pylori – Analysis ; RNA – Genetic Aspects ; RNA – Analysis
    ISSN: 1046-2023
    Source: Cengage Learning, Inc.
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  • 7
    Language: English
    In: The Plant cell, January 2012, Vol.24(1), pp.123-36
    Description: Gene expression in plastids of higher plants is dependent on two different transcription machineries, a plastid-encoded bacterial-type RNA polymerase (PEP) and a nuclear-encoded phage-type RNA polymerase (NEP), which recognize distinct types of promoters. The division of labor between PEP and NEP during plastid development and in mature chloroplasts is unclear due to a lack of comprehensive information on promoter usage. Here, we present a thorough investigation into the distribution of PEP and NEP promoters within the plastid genome of barley (Hordeum vulgare). Using a novel differential RNA sequencing approach, which discriminates between primary and processed transcripts, we obtained a genome-wide map of transcription start sites in plastids of mature first leaves. PEP-lacking plastids of the albostrians mutant allowed for the unambiguous identification of NEP promoters. We observed that the chloroplast genome contains many more promoters than genes. According to our data, most genes (including genes coding for photosynthesis proteins) have both PEP and NEP promoters. We also detected numerous transcription start sites within operons, indicating transcriptional uncoupling of genes in polycistronic gene clusters. Moreover, we mapped many transcription start sites in intergenic regions and opposite to annotated genes, demonstrating the existence of numerous noncoding RNA candidates.
    Keywords: Chloroplasts -- Genetics ; DNA-Directed RNA Polymerases -- Metabolism ; Hordeum -- Enzymology ; Plastids -- Enzymology ; RNA, Untranslated -- Genetics
    ISSN: 10404651
    E-ISSN: 1532-298X
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  • 8
    Language: English
    In: 2013, Vol.9(5), p.e1003495
    Description: Campylobacter jejuni is currently the leading cause of bacterial gastroenteritis in humans. Comparison of multiple Campylobacter strains revealed a high genetic and phenotypic diversity. However, little is known about differences in transcriptome organization, gene expression, and small RNA (sRNA) repertoires. Here we present the first comparative primary transcriptome analysis based on the differential RNA–seq (dRNA–seq) of four C. jejuni isolates. Our approach includes a novel, generic method for the automated annotation of transcriptional start sites (TSS), which allowed us to provide genome-wide promoter maps in the analyzed strains. These global TSS maps are refined through the integration of a SuperGenome approach that allows for a comparative TSS annotation by mapping RNA–seq data of multiple strains into a common coordinate system derived from a whole-genome alignment. Considering the steadily increasing amount of RNA–seq studies, our automated TSS annotation will not only facilitate transcriptome annotation for a wider range of pro- and eukaryotes but can also be adapted for the analysis among different growth or stress conditions. Our comparative dRNA–seq analysis revealed conservation of most TSS, but also single-nucleotide-polymorphisms (SNP) in promoter regions, which lead to strain-specific transcriptional output. Furthermore, we identified strain-specific sRNA repertoires that could contribute to differential gene regulation among strains. In addition, we identified a novel minimal CRISPR-system in Campylobacter of the type-II CRISPR subtype, which relies on the host factor RNase III and a trans-encoded sRNA for maturation of crRNAs. This minimal system of Campylobacter , which seems active in only some strains, employs a unique maturation pathway, since the crRNAs are transcribed from individual promoters in the upstream repeats and thereby minimize the requirements for the maturation machinery. Overall, our study provides new insights into strain-specific transcriptome organization and sRNAs, and reveals genes that could modulate phenotypic variation among strains despite high conservation at the DNA level. ; Many species have evolved into diverse strains with phenotypic and genotypic variations that facilitate adaptation to different ecological niches and, in the case of pathogens, to different hosts. Whereas comparison of genome sequences reveals differences and similarities among strains, the consequences of genomic variations can be tracked by studying the functional output from the genome. RNA sequencing has been revolutionizing transcriptome analyses of both pro- and eukaryotes. However, the bioinformatics-based analysis is still lagging behind, and transcriptome features are often manually annotated, which is laborious and time-consuming. This is even more compounded for the analyses of multiple strains. Here we compared the primary transcriptomes of four isolates of , the leading cause of bacterial gastroenteritis in humans, and provide genome-wide transcriptional start site (TSS) maps using a novel automated annotation method. Our comparative RNA–seq showed that most TSS are conserved in multiple strains, but we also observed SNP–dependent promoter usage. Furthermore, we identified a novel minimal RNA–based CRISPR immune system as well as strain-specific small RNA repertoires. Our automated, comparative TSS annotation will facilitate and improve transcriptome annotation for a wider range of organisms and provides insights into the contribution of transcriptome differences to phenotypic variation among closely related species.
    Keywords: Research Article ; Biology
    ISSN: 1553-7390
    E-ISSN: 1553-7404
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  • 9
    Language: English
    In: Methods, 15 September 2015, Vol.86, pp.89-101
    Description: The global mapping of transcription boundaries is a key step in the elucidation of the full complement of transcriptional features of an organism. It facilitates the annotation of operons and untranslated regions as well as novel transcripts, including - and -encoded small RNAs (sRNAs). So called RNA sequencing (RNA-seq) based on deep sequencing of cDNAs has greatly facilitated transcript mapping with single nucleotide resolution. However, conventional RNA-seq approaches typically cannot distinguish between primary and processed transcripts. Here we describe the recently developed differential RNA-seq (dRNA-seq) approach, which facilitates the annotation of transcriptional start sites (TSS) based on deep sequencing of two differentially treated cDNA library pairs, with one library being enriched for primary transcripts. Using the human pathogen as a model organism, we describe the application of dRNA-seq together with an automated TSS annotation approach for generation of a genome-wide TSS map in bacteria. Besides a description of transcriptome and regulatory features that can be identified by this approach, we discuss the impact of different library preparation protocols and sequencing platforms as well as manual and automated TSS annotation. Moreover, we have set up an easily accessible online browser for visualization of the transcriptome data from this and our previous dRNA-seq study.
    Keywords: Differential RNA-Seq ; Transcriptional Start Sites ; Comparative Transcriptomics ; Small Rnas ; Promoter Motifs ; Gene Regulation ; 5′Utr ; Chemistry ; Anatomy & Physiology
    ISSN: 1046-2023
    E-ISSN: 1095-9130
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  • 10
    Language: English
    In: BMC Genomics, Jan 17, 2012, Vol.13, p.25
    Description: Background Invasion of intestinal epithelial cells by Salmonella enterica serovar Typhimurium (S. Typhimurium) requires expression of the extracellular virulence gene expression programme (ST.sup.EX.sup.), 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 wild-type S. Typhimurium and a ppGpp null strain under growth conditions which model ST.sup.EX.sup.. In doing so we show that ppGpp plays a much wider role in regulating the S. Typhimurium ST.sup.EX .sup.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.
    Keywords: Rna ; Transcription (Genetics) ; Genomes ; Salmonella ; Chromosomes ; Bacterial Genetics ; Gene Expression ; Genomics
    ISSN: 1471-2164
    Source: Cengage Learning, Inc.
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