Dataset Information

Characterization of the transcriptome of Haloferax volcanii, grown under four different conditions, with mixed RNA-Seq.

ABSTRACT: Haloferax volcanii is a well-established model species for haloarchaea. Small scale RNomics and bioinformatics predictions were used to identify small non-coding RNAs (sRNAs), and deletion mutants revealed that sRNAs have important regulatory functions. A recent dRNA-Seq study was used to characterize the primary transcriptome. Unexpectedly, it was revealed that, under optimal conditions, H. volcanii contains more non-coding sRNAs than protein-encoding mRNAs. However, the dRNA-Seq approach did not contain any length information. Therefore, a mixed RNA-Seq approach was used to determine transcript length and to identify additional transcripts, which are not present under optimal conditions. In total, 50 million paired end reads of 150 nt length were obtained. 1861 protein-coding RNAs (cdRNAs) were detected, which encoded 3092 proteins. This nearly doubled the coverage of cdRNAs, compared to the previous dRNA-Seq study. About 2/3 of the cdRNAs were monocistronic, and 1/3 covered more than one gene. In addition, 1635 non-coding sRNAs were identified. The highest fraction of non-coding RNAs were cis antisense RNAs (asRNAs). Analysis of the length distribution revealed that sRNAs have a median length of about 150 nt. Based on the RNA-Seq and dRNA-Seq results, genes were chosen to exemplify characteristics of the H. volcanii transcriptome by Northern blot analyses, e.g. 1) the transcript patterns of gene clusters can be straightforward, but also very complex, 2) many transcripts differ in expression level under the four analyzed conditions, 3) some genes are transcribed into RNA isoforms of different length, which can be differentially regulated, 4) transcripts with very long 5'-UTRs and with very long 3'-UTRs exist, and 5) about 30% of all cdRNAs have overlapping 3'-ends, which indicates, together with the asRNAs, that H. volcanii makes ample use of sense-antisense interactions. Taken together, this RNA-Seq study, together with a previous dRNA-Seq study, enabled an unprecedented view on the H. volcanii transcriptome.

SUBMITTER: Laass S

PROVIDER: S-EPMC6490895 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Characterization of the transcriptome of Haloferax volcanii, grown under four different conditions, with mixed RNA-Seq.

Laass Sebastian S Monzon Vivian A VA Kliemt Jana J Hammelmann Matthias M Pfeiffer Friedhelm F Förstner Konrad U KU Soppa Jörg J

PloS one 20190430 4

Haloferax volcanii is a well-established model species for haloarchaea. Small scale RNomics and bioinformatics predictions were used to identify small non-coding RNAs (sRNAs), and deletion mutants revealed that sRNAs have important regulatory functions. A recent dRNA-Seq study was used to characterize the primary transcriptome. Unexpectedly, it was revealed that, under optimal conditions, H. volcanii contains more non-coding sRNAs than protein-encoding mRNAs. However, the dRNA-Seq approach did n ...[more]

PMID: 31039177

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Project description:In this study the transcriptome of Haloferax volcanii DS70 (Wendoloski D, Ferrer C, Dyall-Smith ML. Microbiology. 2001, 147: 959-64), grown under of a variety of conditions, was mapped using a custom genome-wide tiled array consisting of 25 nt probes spaced in 35 nt windows. H. volcanii is a facultative aerobe capable of growth on simple carbon and nitrogen sources, it is amenable to genetic manipulations and has become a widely used model organism for studies on haloarchaeal physiology and global gene regulation. Of particular interest is the identification of genes encoding enzymes needed for growth on simple carbon and nitrogen sources and how these cells respond to limitations in these key nutrients. To address these questions we have compared the RNA populations of cells undergoing balanced growth in completely defined minimal medium (CDM) with succinate-glycerol, pyruvate or lactate as sole carbon sources with RNA from cells grown in complex rich medium (CX) containing yeast extract and tryptone. Data from these studies have identified a core group of genes expressing during balanced growth in either condition as well as specific genes associated with the biosynthesis of amino acids, nucleotides and other key building blocks. The response to nutrient limitation was also investigated. RNA populations from cells entering stationary phase from growth in CDM and CX media were compared to those undergoing balanced growth, and these RNAs were also compared with RNA populations of cells subjected to nitrogen or carbon starvation. Growth in the presence of histidine as the sole source of nitrogen in CDM medium also presented a model for growth with a “poor” nitrogen source. The results of these studies identified specific subpopulations of genes associated with nitrogen limitation and general nutrient limitation associated with the transition to stationary phase. Many of the responsive protein encoding genes were assigned general functions based on their similarity to known proteins; however, in each comparison, approximately half of the affected genes encode proteins classified as unknown or hypothetical proteins. The haloarchaea are also distinct among the Archaea in having multiple genes encoding the general transcription factor proteins TATA binding protein (TBP) and transcription factor IIB (TFB). We observed differential expression of these genes, providing further support for the proposal that alternative TBP-TFB pairings are associated with programmed changes in gene expression in the haloarchaea. RNA was prepared from H. volcanii DS70 cells grown under a variety of conditions. These include: balanced growth in completely defined medium with succinate and glycerol, pyruvate, or lactate as sole carbon source; balanced growth in complex medium with yeast extract and tryptone; starvation for nitrogen or carbon by re-suspending cells, undergoing balanced growth, in media lacking these agents; balanced growth in the presence of low (10% w/v NaCl) and high (20 % or 25% w/v NaCl) salt, and cells entering stationary from growth in CDM or CX media. Four independent cultures were prepared for each growth variable and RNA was isolated from each culture. RNA samples were identified as acceptable for use in array analysis if clear 23S rRNA, 16S rRNA and tRNA-sized bands were present in agarose gel and capillary electrophoresis analysis of the samples. Three RNA samples were chosen for each growth variable and each was used as the source material for a separate chip experiment; these constituted three biological replicates for the growth variable. Data from 48 chip hybridizations, representing 16 growth conditions, were combined for the quantile scaling (Auer H, Newsom DL, Nowak NJ, McHugh KM, Singh S, Yu CY, Yang Y, Wenger GD, Gastier-Foster JM, Kornacker K. BMC Genomics. 2007, 8:111-124) presented in the data analyses. Two additional experiments are included where the RNAs of three independent biological samples were pooled into a single RNA population and used as the source material for an array experiment. These included RNA samples enriched for species less than 500 nt that were isolated from cells undergoing balanced growing, and entry into stationary phase, in CDM or CX media. A second pooled series were obtained from the H. volcanii DS70 mutant strains ASD40 (∆pyrF) and ASD41 (∆pyrF∆hutR) grown in CDM with ammonium ion or histidine as the sole source of nitrogen. Pooled samples are presented as quantile normalized values (Bolstad, B.M., Irizarry, R.A., Astrand, M. and Speed, T.P. Bioinformatics. 2003, 19: 185-193). Total genomic DNA from H. volcanii DS70 was also used as probe, in two independent array experiments, to measure the inherent hybridization to the probes. These data are presented as quantile scaled values (Auer et. al., 2007).

Dataset Information

Characterization of the transcriptome of Haloferax volcanii, grown under four different conditions, with mixed RNA-Seq.

Publications

Characterization of the transcriptome of Haloferax volcanii, grown under four different conditions, with mixed RNA-Seq.

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