Project description:The non-coding transcriptome of the hyperthermophilic archaeon Pyrococcus abyssi is investigated using the RNA-seq technology. A dedicated computational pipeline analyzes RNA-seq reads and prior genome annotation to identify small RNAs, untranslated regions of mRNAs, and cis-encoded antisense transcripts. Unlike other archaea, such as Sulfolobus and Halobacteriales, P. abyssi produces few leaderless mRNA transcripts. Antisense transcription is widespread (215 transcripts) and targets protein-coding genes that are less conserved than average genes. We identify at least three novel H/ACA-like guide RNAs among the newly characterized non-coding RNAs. Long 5' UTRs in mRNAs of ribosomal proteins and amino-acid biosynthesis genes strongly suggest the presence of cis-regulatory leaders in these mRNAs. We selected a high-interest subset of non-coding RNAs based on their strong promoters, high GC-content, phylogenetic conservation, or abundance. Some of the novel small RNAs and long 5' UTRs display high GC contents, suggesting unknown structural RNA functions. However, we were surprised to observe that most of the high-interest RNAs are AU-rich, which suggests an absence of stable secondary structure in the high-temperature environment of P. abyssi. Yet, these transcripts display other hallmarks of functionality, such as high expression or high conservation, which leads us to consider possible RNA functions that do not require extensive secondary structure.
Project description:The non-coding transcriptome of the hyperthermophilic archaeon Pyrococcus abyssi is investigated using the RNA-seq technology. A dedicated computational pipeline analyzes RNA-seq reads and prior genome annotation to identify small RNAs, untranslated regions of mRNAs, and cis-encoded antisense transcripts. Unlike other archaea, such as Sulfolobus and Halobacteriales, P. abyssi produces few leaderless mRNA transcripts. Antisense transcription is widespread (215 transcripts) and targets protein-coding genes that are less conserved than average genes. We identify at least three novel H/ACA-like guide RNAs among the newly characterized non-coding RNAs. Long 5' UTRs in mRNAs of ribosomal proteins and amino-acid biosynthesis genes strongly suggest the presence of cis-regulatory leaders in these mRNAs. We selected a high-interest subset of non-coding RNAs based on their strong promoters, high GC-content, phylogenetic conservation, or abundance. Some of the novel small RNAs and long 5' UTRs display high GC contents, suggesting unknown structural RNA functions. However, we were surprised to observe that most of the high-interest RNAs are AU-rich, which suggests an absence of stable secondary structure in the high-temperature environment of P. abyssi. Yet, these transcripts display other hallmarks of functionality, such as high expression or high conservation, which leads us to consider possible RNA functions that do not require extensive secondary structure. directional RNA-seq, Illumina GA-IIx
Project description:Recent advances in the study of archaeal DNA replication have uncovered defined replication origins (oriC) and demonstrated specific binding of the Cdc6/Orc1 protein and Mcm helicase to oriC in vivo and/or in vitro. The oriC of the hyperthermophilic archaeon Pyrococcus abyssi is characterized by 13 bp repeats, AT-rich regions and an inverted repeat whose precise roles remain unclear. We report here that the 13 bp repeats are widespread in the three Pyrococcus genomes. Nevertheless, by means of chromatin immunoprecipitation coupled with hybridization on a whole genome microarray (ChIP-chip), we found that binding of P. abyssi Cdc6/Orc1 to oriC in vivo was highly specific both in exponential and stationary phases, allowing oriC to be distinguished in the 1.8 M bp genome. ChIP-chip analysis also indicated that a single 13 bp repeat is not sufficient for stable binding of Cdc6/Orc1. Purified Cdc6/Orc1 binds a DNA fragment containing the inverted repeat of oriC with a relatively low affinity, suggesting that multiple clusters of the 13 bp repeat discovered in this study contribute to the stable binding of Cdc6/Orc1 to oriC. Our ChIP-chip analysis revealed that Mcm also binds oriC only in proliferating cells, consistent with its role as a licensing factor. Finally, we found that both Cdc6/Orc1 and Mcm have one additional target site. Notably, Mcm binds constitutively to a GC-rich region containing two rRNA genes and a tRNA gene, suggesting a role of archaeal Mcm in DNA replication and/or transcription of this peculiar region. Keywords: ChIP-chip
Project description:We designed an experimental setup to investigate the transcriptomic and proteomic responses of the hyperthermophilic archaeon Pyrococcus furiosus to heat and cold shock. P. furiosus is a model organism for studying microbial adaptation to extreme environments, including deep-sea hydrothermal vents with temperature gradients ranging from 1°C to 400°C. We aimed to simulate critical conditions where P. furiosus cannot grow and to examine the immediate response to thermal stress as well as the recovery process.
Project description:Oxidative Stress Protection and the Repair Response To Hydrogen Peroxide in the Hyperthermophilic Archaeon Pyrococcus furiosus Pyrococcus furiosus is a shallow marine, anaerobic archaeon that grows optimally at 100°C. Addition of H2O2 (0.5 mM) to a growing culture resulted in cessation of growth with a 2 hour lag before normal growth resumed. Whole genome transcriptional profiling revealed that the main response occurs within 30 min of peroxide addition, with the up-regulation of 62 open reading frames (ORFs), 36 of which are part of 10 potential operons. More than half of the up-regulated ORFs are of unknown function while some others encode proteins that are involved potentially in sequestering iron and sulfide, in DNA repair and in generating NADPH. This response is thought to involve primarily damage repair rather than protection, since cultures exposed to sub-toxic levels of H2O2 were not more resistant to the subsequent addition of H2O2 (0.5 – 5.0 mM). Consequently, there is little if any induced protective response to peroxide, rather, the organism maintains a constitutive protective mechanism involving high levels of oxidoreductase-type enzymes such as superoxide reductase, rubrerythrin and alkyl hydroperoxide reductase I. The related hyperthermophiles P. woesei and Thermococcus kodakaraensis were more sensitive to peroxide than P. furiosus, apparently due to the lack of several of its peroxide-responsive ORFs.
Project description:Recombinant aRNase J from P. abyssi, with C-terminal (His)6-tag (aRNase J-His), was produced, purified and used as baits for AP-MS experiments. In order to discriminate specific protein interaction from column background, we performed mock AP-MS analyses with P. abyssi cell extracts in absence of protein bait. In addition, to determine if some interactions are mediated through RNA or DNA, we implemented a nuclease treatment after incubating the cell extract with the bait. The co-purified partners were identified by bottom-up proteomic techniques coupled with mass spectrometry.