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  • Article  (12)
  • Sharma, Cynthia M.  (12)
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  • 1
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 23 November 2010, Vol.107(47), pp.20435-40
    Description: The abundant class of bacterial Hfq-associated small regulatory RNAs (sRNAs) parallels animal microRNAs in their ability to control multiple genes at the posttranscriptional level by short and imperfect base pairing. In contrast to the universal length and seed pairing mechanism of microRNAs, the sRNAs are heterogeneous in size and structure, and how they regulate multiple targets is not well understood. This paper provides evidence that a 5' located sRNA domain is a critical element for the control of a large posttranscriptional regulon. We show that the conserved 5' end of RybB sRNA recognizes multiple mRNAs of Salmonella outer membrane proteins by ≥7-bp Watson-Crick pairing. When fused to an unrelated sRNA, the 5' domain is sufficient to guide target mRNA degradation and maintain σ(E)-dependent envelope homeostasis. RybB sites in mRNAs are often conserved and flanked by 3' adenosine. They are found in a wide sequence window ranging from the upstream untranslated region to the deep coding sequence, indicating that some targets might be repressed at the level of translation, whereas others are repressed primarily by mRNA destabilization. Autonomous 5' domains seem more common in sRNAs than appreciated and might improve the design of synthetic RNA regulators.
    Keywords: Bacterial Outer Membrane Proteins -- Metabolism ; Gene Expression Regulation, Bacterial -- Genetics ; RNA, Messenger -- Metabolism ; Regulatory Sequences, Ribonucleic Acid -- Genetics ; Regulon -- Genetics ; Salmonella -- Genetics
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 2
    In: Nature, 2011, Vol.471(7340), p.602
    Description: CRISPR/Cas systems constitute a widespread class of immunity systems that protect bacteria and archaea against phages and plasmids, and commonly use repeat/spacer-derived short crRNAs to silence foreign nucleic acids in a sequence-specific manner. Although the maturation of crRNAs represents a key event in CRISPR activation, the responsible endoribonucleases (CasE, Cas6, Csy4) are missing in many CRISPR/Cas subtypes. Here, differential RNA sequencing of the human pathogen Streptococcus pyogenes uncovered tracrRNA, a trans -encoded small RNA with 24 nucleotide complementarity to the repeat regions of crRNA precursor transcripts. We show that tracrRNA directs the maturation of crRNAs by the activities of the widely conserved endogenous RNase III and the CRISPR-associated Csn1 protein; all these components are essential to protect S. pyogenes against prophage-derived DNA. Our study reveals a novel pathway of small guide RNA maturation and the first example of a host factor (RNase III) required for bacterial RNA-mediated immunity against invaders.
    Keywords: Article;
    ISSN: 0028-0836
    E-ISSN: 14764687
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  • 3
    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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  • 4
    In: Molecular Microbiology, September 2011, Vol.81(5), pp.1144-1165
    Description: GcvB is one of the most highly conserved Hfq‐associated small RNAs in Gram‐negative bacteria and was previously reported to repress several ABC transporters for amino acids. To determine the full extent of GcvB‐mediated regulation in , we combined a genome‐wide experimental approach with biocomputational target prediction. Comparative pulse expression of wild‐type versus mutant sRNA variants revealed that GcvB governs a large post‐transcriptional regulon, impacting ∼1% of all genes via its conserved G/U‐rich domain R1. Complementary predictions of C/A‐rich binding sites in mRNAs and reporter fusion experiments increased the number of validated GcvB targets to more than 20, and doubled the number of regulated amino acid transporters. Unlike the previously described targeting via the single R1 domain, GcvB represses the glycine transporter CycA by exceptionally redundant base‐pairing. This novel ability of GcvB is focused upon the one target that could feedback‐regulate the glycine‐responsive synthesis of GcvB. Several newly discovered mRNA targets involved in amino acid metabolism, including the global regulator Lrp, question the previous assumption that GcvB simply acts to limit unnecessary amino acid uptake. Rather, GcvB rewires primary transcriptional control circuits and seems to act as a distinct regulatory node in amino acid metabolism.
    Keywords: Glycine -- Physiological Aspects ; Genetic Research -- Physiological Aspects ; Genomics -- Physiological Aspects ; Messenger Rna -- Physiological Aspects;
    ISSN: 0950-382X
    E-ISSN: 1365-2958
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  • 5
    Language: English
    In: Current Opinion in Microbiology, June 2014, Vol.19, pp.97-105
    Description: RNA-sequencing has revolutionized the quantitative and qualitative analysis of transcriptomes in both prokaryotes and eukaryotes. It provides a generic approach for gene expression profiling, annotation of transcript boundaries and operons, as well as identifying novel transcripts including small noncoding RNA molecules and antisense RNAs. We recently developed a differential RNA-seq (dRNA-seq) method which in addition to the above, yields information as to whether a given RNA is a primary or processed transcript. Originally applied to describe the primary transcriptome of the gastric pathogen , dRNA-seq has since provided global maps of transcriptional start sites in diverse species, informed new biology in the CRISPR-Cas9 system, advanced to a tool for comparative transcriptomics, and inspired simultaneous RNA-seq of pathogen and host.
    Keywords: Biology
    ISSN: 1369-5274
    E-ISSN: 1879-0364
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  • 6
    Language: English
    In: PLoS Genetics, 2012, Vol.8(6), p.e1002782
    Description: 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. ; Control of mRNA stability is crucial for bacteria to survive and rapidly adapt to environmental changes and stress conditions. The molecular players and the degradation pathways involved in these adaptive processes are poorly understood in . The universally conserved double-strand-specific endoribonuclease III (RNase III) in is known to repress the synthesis of several virulence factors and was recently implicated in genome-wide mRNA processing mediated by antisense transcripts. We present here the first global map of direct RNase III targets in . Deep sequencing was used to identify RNAs associated with epitope-tagged wild-type RNase III and two catalytically impaired but binding-competent mutant proteins . Experimental validation revealed an unexpected variety of structured RNA transcripts as novel RNase III substrates. In addition to rRNA operon maturation, autoregulation, degradation of structured RNAs, and antisense regulation, we propose novel mechanisms by which RNase III increases mRNA translation. Overall, this study shows that RNase III has a broad function in gene regulation of . We can now address more specifically the roles of this universally conserved enzyme in gene regulation in response to stress and during host infection.
    Keywords: Research Article ; Biology ; Genetics And Genomics ; Microbiology
    ISSN: 1553-7390
    E-ISSN: 1553-7404
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  • 7
    In: Molecular Microbiology, December 2009, Vol.74(6), pp.1497-1512
    Description: Exposure to oxygen and light generates photooxidative stress by the bacteriochlorophyll mediated formation of singlet oxygen (O) in . Our study reports the genome‐wide search for small RNAs (sRNAs) involved in the regulatory response to O. By using 454 pyrosequencing and Northern blot analysis, we identified 20 sRNAs from aerobic cultures or following treatment with O or superoxide (O). One sRNA was specifically induced by O and its expression depends on the extracytoplasmic function sigma factor RpoE. Two sRNAs induced by O and O were cotranscribed with upstream genes preceded by promoters with target sequences for the alternative sigma factors RpoH and RpoH. The most abundant sRNA was processed in the presence of O but not by O. From this and a second sRNA a conserved 3′‐segment accumulated from a larger precursor. Absence of the RNA chaperone Hfq changed the half‐lives, abundance and processing of O‐affected sRNAs. Orthologues of three sRNA genes are present in different alpha‐proteobacteria, but the majority was unique to or species. Our discovery that abundant sRNAs are affected by O exposure extends the knowledge on the role of sRNAs and Hfq in the regulatory response to oxidative stress.
    Keywords: Active Oxygen -- Analysis ; Genomics -- Analysis ; Molecular Chaperones -- Analysis ; Rna -- Analysis ; Superoxides -- Analysis;
    ISSN: 0950-382X
    E-ISSN: 1365-2958
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  • 8
    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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  • 9
    Language: English
    In: Molecular microbiology, June 2011, Vol.80(6), pp.1479-95
    Description: The photosynthetic alphaproteobacterium Rhodobacter sphaeroides has to cope with photooxidative stress that is caused by the bacteriochlorophyll a-mediated formation of singlet oxygen ((1)O(2)). Exposure to (1)O(2) induces the alternative sigma factors RpoE and RpoH(II) which then promote transcription of photooxidative stress-related genes, including small RNAs (sRNAs). The ubiquitous RNA chaperone Hfq is well established to interact with and facilitate the base-pairing of sRNAs and target mRNAs to influence mRNA stability and/or translation. Here we report on the pleiotropic phenotype of a Δhfq mutant of R. sphaeroides, which is less pigmented, produces minicells and is more sensitive to (1)O(2). The higher (1)O(2) sensitivity of the Δhfq mutant is paralleled by a reduced RpoE activity and a disordered induction of RpoH(II)-dependent genes. We used co-immunoprecipitation of FLAG-tagged Hfq combined with RNA-seq to identify association of at least 25 sRNAs and of mRNAs encoding cell division proteins and ribosomal proteins with Hfq. Remarkably, 〉 70% of the Hfq-bound sRNAs are (1)O(2)-affected. Proteomics analysis of the Hfq-deficient strain revealed an impact of Hfq on amino acid transport and metabolic functions. Our data demonstrate for the first time an involvement of Hfq in regulation of photosynthesis genes and in the photooxidative stress response.
    Keywords: Gene Expression Regulation, Bacterial ; Oxidative Stress ; Protein Binding ; Bacterial Proteins -- Metabolism ; Host Factor 1 Protein -- Metabolism ; Rhodobacter Sphaeroides -- Metabolism
    ISSN: 0950382X
    E-ISSN: 1365-2958
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  • 10
    Language: English
    In: PLoS Computational Biology, 2009, Vol.5(9), p.e1000502
    Description: With few exceptions, current methods for short read mapping make use of simple seed heuristics to speed up the search. Most of the underlying matching models neglect the necessity to allow not only mismatches, but also insertions and deletions. Current evaluations indicate, however, that very different error models apply to the novel high-throughput sequencing methods. While the most frequent error-type in Illumina reads are mismatches, reads produced by 454's GS FLX predominantly contain insertions and deletions (indels). Even though 454 sequencers are able to produce longer reads, the method is frequently applied to small RNA (miRNA and siRNA) sequencing. Fast and accurate matching in particular of short reads with diverse errors is therefore a pressing practical problem. We introduce a matching model for short reads that can, besides mismatches, also cope with indels. It addresses different error models. For example, it can handle the problem of leading and trailing contaminations caused by primers and poly-A tails in transcriptomics or the length-dependent increase of error rates. In these contexts, it thus simplifies the tedious and error-prone trimming step. For efficient searches, our method utilizes index structures in the form of enhanced suffix arrays. In a comparison with current methods for short read mapping, the presented approach shows significantly increased performance not only for 454 reads, but also for Illumina reads. Our approach is implemented in the software segemehl available at http://www.bioinf.uni-leipzig.de/Software/segemehl/ . ; The successful mapping of high-throughput sequencing (HTS) reads to reference genomes largely depends on the accuracy of both the sequencing technologies and reference genomes. Current mapping algorithms focus on mapping with mismatches but largely neglect insertions and deletions—regardless of whether they are caused by sequencing errors or genomic variation. Furthermore, trailing contaminations by primers and declining read qualities can be cumbersome for programs that allow a maximum number of mismatches. We have developed and implemented a new approach for short read mapping that, in a first step, computes exact matches of the read and the reference genome. The exact matches are then modified by a limited number of mismatches, insertions and deletions. From the set of exact and inexact matches, we select those with minimum score-based E-values. This gives a set of regions in the reference genome which is aligned to the read using Myers bitvector algorithm . Our method utilizes enhanced suffix arrays to quickly find the exact and inexact matches. It maps more reads and achieves higher recall rates than previous methods. This consistently holds for reads produced by 454 as well as Illumina sequencing technologies.
    Keywords: Research Article ; Computational Biology ; Computational Biology -- Genomics ; Genetics And Genomics -- Bioinformatics
    ISSN: 1553-734X
    E-ISSN: 1553-7358
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