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  • 1
    In: PLoS ONE, 2014, Vol.9(8)
    Description: CRISPR-Cas systems provide immunity against viral attacks in archaeal and bacterial cells. Type I systems employ a Cas protein complex termed Cascade, which utilizes small CRISPR RNAs to detect and degrade the exogenic DNA. A small sequence motif, the PAM, marks the foreign substrates. Previously, a recombinant type I-A Cascade complex from the archaeon Thermoproteus tenax was shown to target and degrade DNA in vitro, dependent on a native PAM sequence. Here, we present the biochemical analysis of the small subunit, Csa5, of this Cascade complex. T. tenax Csa5 preferentially bound ssDNA and mutants that showed decreased ssDNA-binding and reduced Cascade-mediated DNA cleavage were identified. Csa5 oligomerization prevented DNA binding. Specific recognition of the PAM sequence was not observed. Phylogenetic analyses identified Csa5 as a universal member of type I-A systems and revealed three distinct groups. A potential role of Csa5 in R-loop stabilization is discussed.
    Keywords: Research Article ; Biology And Life Sciences
    E-ISSN: 1932-6203
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  • 2
    Language: English
    In: PLoS ONE, August 22, 2014, Vol.9(8)
    Description: CRISPR-Cas systems provide immunity against viral attacks in archaeal and bacterial cells. Type I systems employ a Cas protein complex termed Cascade, which utilizes small CRISPR RNAs to detect and degrade the exogenic DNA. A small sequence motif, the PAM, marks the foreign substrates. Previously, a recombinant type I-A Cascade complex from the archaeon Thermoproteus tenax was shown to target and degrade DNA in vitro, dependent on a native PAM sequence. Here, we present the biochemical analysis of the small subunit, Csa5, of this Cascade complex. T. tenax Csa5 preferentially bound ssDNA and mutants that showed decreased ssDNA-binding and reduced Cascade-mediated DNA cleavage were identified. Csa5 oligomerization prevented DNA binding. Specific recognition of the PAM sequence was not observed. Phylogenetic analyses identified Csa5 as a universal member of type I-A systems and revealed three distinct groups. A potential role of Csa5 in R-loop stabilization is discussed.
    Keywords: Oligomers – Chemical Properties ; Oligomers – Analysis ; DNA Binding – Chemical Properties ; DNA Binding – Analysis ; Phylogeny – Chemical Properties ; Phylogeny – Analysis ; DNA – Chemical Properties ; DNA – Analysis
    ISSN: 1932-6203
    Source: Cengage Learning, Inc.
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  • 3
    Language: English
    In: Nucleic acids research, April 2014, Vol.42(8), pp.5125-38
    Description: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) systems of type I use a Cas ribonucleoprotein complex for antiviral defense (Cascade) to mediate the targeting and degradation of foreign DNA. To address molecular features of the archaeal type I-A Cascade interference mechanism, we established the in vitro assembly of the Thermoproteus tenax Cascade from six recombinant Cas proteins, synthetic CRISPR RNAs (crRNAs) and target DNA fragments. RNA-Seq analyses revealed the processing pattern of crRNAs from seven T. tenax CRISPR arrays. Synthetic crRNA transcripts were matured by hammerhead ribozyme cleavage. The assembly of type I-A Cascade indicates that Cas3' and Cas3'' are an integral part of the complex, and the interference activity was shown to be dependent on the crRNA and the matching target DNA. The reconstituted Cascade was used to identify sequence motifs that are required for efficient DNA degradation and to investigate the role of the subunits Cas7 and Cas3'' in the interplay with other Cascade subunits.
    Keywords: Crispr-Cas Systems ; Archaeal Proteins -- Metabolism ; Crispr-Associated Proteins -- Metabolism ; Deoxyribonucleases -- Metabolism
    ISSN: 03051048
    E-ISSN: 1362-4962
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  • 4
    Language: German
    In: BIOspektrum, 2014, Vol.20(6), pp.615-617
    Description: Hyperthermophilic archaea need to adapt to their permanently hot habitats. RNA-Seq analyses were applied to obtain insights into small RNA synthesis, maturation, and stability at extreme growth temperatures. Modifications, including methylation and circularization, are used to stabilize RNA molecules. Antiviral measures are abundant in hyperthermophilic archaea which might include the frequently occurring fragmentation of tRNA genes.
    Keywords: Life Sciences ; Life Sciences, General ; Biochemistry, General ; Human Genetics ; Microbiology ; Developmental Biology ; Pharmacology/Toxicology ; Sciences (General);
    ISSN: 0947-0867
    E-ISSN: 1868-6249
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  • 5
    In: mBio, 2017, Vol.8(4)
    Description: ABSTRACT L7Ae is a universal archaeal protein that recognizes and stabilizes kink-turn (k-turn) motifs in RNA substrates. These structural motifs are widespread in nature and are found in many functional RNA species, including ribosomal RNAs. Synthetic biology approaches utilize L7Ae/k-turn interactions to control gene expression in eukaryotes. Here, we present results of comprehensive RNA immunoprecipitation sequencing (RIP-Seq) analysis of genomically tagged L7Ae from the hyperthermophilic archaeon Sulfolobus acidocaldarius . A large set of interacting noncoding RNAs was identified. In addition, several mRNAs, including the l7ae transcript, were found to contain k-turn motifs that facilitate L7Ae binding. In vivo studies showed that L7Ae autoregulates the translation of its mRNA by binding to a k-turn motif present in the 5′ untranslated region (UTR). A green fluorescent protein (GFP) reporter system was established in Escherichia coli and verified conservation of L7Ae-mediated feedback regulation in Archaea . Mobility shift assays confirmed binding to a k-turn in the transcript of nop5-fibrillarin , suggesting that the expression of all C/D box sRNP core proteins is regulated by L7Ae. These studies revealed that L7Ae-mediated gene regulation evolved in archaeal organisms, generating new tools for the modulation of synthetic gene circuits in bacteria. IMPORTANCE L7Ae is an essential archaeal protein that is known to structure ribosomal RNAs and small RNAs (sRNAs) by binding to their kink-turn motifs. Here, we utilized RIP-Seq methodology to achieve a first global analysis of RNA substrates for L7Ae. Several novel interactions with noncoding RNA molecules (e.g., with the universal signal recognition particle RNA) were discovered. In addition, L7Ae was found to bind to mRNAs, including its own transcript’s 5′ untranslated region. This feedback-loop control is conserved in most archaea and was incorporated into a reporter system that was utilized to control gene expression in bacteria. These results demonstrate that L7Ae-mediated gene regulation evolved originally in archaeal organisms. The feedback-controlled reporter gene system can easily be adapted for synthetic biology approaches that require strict gene expression control.
    Keywords: Research Article ; Archaea ; Rna Binding Proteins ; Rna Structure ; Gene Regulation
    E-ISSN: 2150-7511
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  • 6
    Language: English
    In: eLife, 24 October 2015, Vol.4
    Description: Signal recognition particles (SRPs) are universal ribonucleoprotein complexes found in all three domains of life that direct the cellular traffic and secretion of proteins. These complexes consist of SRP proteins and a single, highly structured SRP RNA. Canonical SRP RNA genes have not been identified for some Thermoproteus species even though they contain SRP19 and SRP54 proteins. Here, we show that genome rearrangement events in Thermoproteus tenax created a permuted SRP RNA gene. The 5'- and 3'-termini of this SRP RNA are located close to a functionally important loop present in all known SRP RNAs. RNA-Seq analyses revealed that these termini are ligated together to generate circular SRP RNA molecules that can bind to SRP19 and SRP54. The circularization site is processed by the tRNA splicing endonuclease. This moonlighting activity of the tRNA splicing machinery permits the permutation of the SRP RNA and creates highly stable and functional circular RNA molecules.
    Keywords: RNA Processing ; Thermoproteus Tenax ; Archaea ; Biochemistry ; Evolutionary Biology ; Genomics ; Signal Recognition Particle ; Splicing ; RNA -- Metabolism ; Signal Recognition Particle -- Metabolism ; Thermoproteus -- Genetics
    E-ISSN: 2050-084X
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  • 7
    Language: English
    In: Methods in enzymology, 2018, Vol.612, pp.413-442
    Description: Noncoding RNAs (ncRNAs) fulfill essential functions in eukaryotes and bacteria, but also in the third domain of life, the Archaea. Many archaeal organisms live in hostile environments that provide unique challenges for their transcriptional and translational regulatory pathways. Computational analyses and RNA-sequencing methodologies allowed for the genome-wide detection of ncRNA molecules in archaea. Several new classes of ncRNAs have been discovered and are expected to enable life in these extreme habitats. Here, we provide an overview of the current knowledge on archaeal ncRNAs and their deduced or biochemically verified functions. In addition, details of applying RNA-seq methodology for the detection of ncRNAs in Sulfolobus acidocaldarius are provided. Identified ncRNAs include small RNAs (sRNAs) that regulate gene expression and C/D box sRNAs that guide 2'-O methylation of target RNAs.
    Keywords: Classification of Archaeal Ncrnas ; Identification of Ncrnas ; RNA Sequencing ; Ncrna ; Sno-Like Guide Srna
    ISSN: 00766879
    E-ISSN: 1557-7988
    Source: MEDLINE/PubMed (U.S. National Library of Medicine)
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  • 8
    Language: English
    In: Annual Review of Genetics, 2018, Vol.52, p.465(23)
    Description: Advances in genome-wide sequence technologies allow for detailed insights into the complexity of RNA landscapes of organisms from all three domains of life. Recent analyses of archaeal transcriptomes identified interaction and regulation networks of noncoding RNAs in this understudied domain. Here, we review current knowledge of small, noncoding RNAs with important functions for the archaeal lifestyle, which often requires adaptation to extreme environments. One focus is RNA metabolism at elevated temperatures in hyperthermophilic archaea, which reveals elevated amounts of RNA-guided RNA modification and virus defense strategies. Genome rearrangement events result in unique fragmentation patterns of noncoding RNA genes that require elaborate maturation pathways to yield functional transcripts. RNA-binding proteins, e.g., L7Ae and LSm, are important for many posttranscriptional control functions of RNA molecules in archaeal cells. We also discuss recent insights into the regulatory potential of their noncoding RNA partners.
    Keywords: Transfer RNA ; Protein Binding ; Antisense RNA ; Genomics
    ISSN: 0066-4197
    E-ISSN: 15452948
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  • 9
    In: Journal of Shellfish Research, 2006, Vol.25(1), p.239-247
    Description: Abstract Germlings of the green alga Ulva sp. were developed as a diet for juvenile Haliotis laevigata (≥3.5 mm shell length) and compared with a current commercial diet consisting of Ulvella lens plus the diatom species Navicula cf. jeffreyi. The utilization of macroalgae germlings (juvenile gametophyte and sporophyte) allowed 3-dimensional growth and subsequently provided greater feed biomass in comparison with the current 2-dimensional commercial feed for the later nursery phase consisting of 5–10 mm (shell length) juvenile abalone. The juvenile abalone showed active feeding on both the Ulva germling diet and the current commercial diet. The Ulvella lens/Navicula cf. jeffreyi diet resulted in abalone of significantly larger shell length at the end of the 14-wk feeding trial. However, the Ulva germling diet recorded significantly larger abalone for the first 4–5 wk, whereas the commercial diet produced significantly larger abalone from week 6 to the end of the trial. The growth rate on both diets exceeded 100 μm.day−1 and the specific growth rates were maintained above 1%.day−1 for the duration of the feeding trial with neither measure portraying significant differences between diets. There was no significant difference in juvenile abalone mortality feeding on the two diets. The Ulva germling consumption exhibited a spike (500 germling blades.abalone−1.day−1) in consumption at week three then, once reduced, a gradual increase occurred until the end of the trial. Ulvella lens consumption demonstrated a similar pattern to Ulva germlings consumption and was significantly, positively correlated. Consumption rates for the two green algae both correlated with juvenile abalone growth. The diatom (Navicula cf. jeffreyi) consumption was affected by plate rotation (light intensity and grazing pressure) rather than juvenile abalone.
    Keywords: Juvenile abalone ; 〈kwd〉haliotis laevigata ; 〈kwd〉ulva ; Germlings ; 〈kwd〉ulvella lens ; Diatoms ; Dietary value
    ISSN: 0730-8000
    E-ISSN: 19436319
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  • 10
    In: Journal of Shellfish Research, 2007, Vol.26(3), p.737-744
    Description: Abstract A variety of red algal species have been identified as potential food sources for juvenile Greenlip Abalone, Haliotis laevigata (〉5 mm shell length). To provide the red algal species in a diet suitable for juvenile abalone three propagation methods; spore production, protoplast isolation, and fragment culture were investigated. The potential algae requirements and consumable costs for each propagation method were determined, using experimental data and values from the literature, to assess the viability of utilizing each of these propagation methods in a commercial abalone nursery. The use of red algal spores required 592–52,000 kg of algae, depending on the level of spore release and the percentage of fertile algal thalli collected. Protoplast isolation reduced the amount of algal biomass to 8.55–910 kg but was affected by the efficiency of the isolation procedure. Even at an efficient production of 1 × 108 protoplasts·g−1 wet weight alga the cost of consumables (enzymes) was $US 13,576. A feeding trial utilizing Laurencia sp. fragments adhered to the plates using agar, produced juvenile abalone growth rates comparable to those obtained with the current commercial nursery diet of the green alga Ulvella lens plus the diatom Navicula cf. jeffreyi. The Laurencia fragments did not regenerate on the plates so it was reapplied weekly, which is not feasible on a commercial scale because it would require 10.6 t of Laurencia and 443 kg of agar at a cost of $US 34,899. Gracilaria sp. fragments were able to regenerate with a growth rate of 4.42%·day−1 and therefore the algal fragments would only need to be applied to the PVC plates once, at the start of the later nursery phase (5 mm SL), reducing the amount of algal biomass to 432 kg. We therefore conclude that regenerating fragment culture (fragments 〈1 mm) is the only method that could successfully produce red algal diets for juvenile abalone on a commercial scale in the later nursery phase.
    Keywords: Juvenile abalone ; 〈kwd〉 haliotis laevigata ; Red algae ; Spore formation ; Protoplast isolation ; Fragment culture ; Algal biomass
    ISSN: 0730-8000
    E-ISSN: 19436319
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