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.

Exploring prokaryotic transcription, operon structures, rRNA maturation and modifications using Nanopore-based native RNA sequencing

“…Quantification by comparison with the NOTEX wildtype set confirms the previous detection of this rRNA variant in a gel-electrophoretic analysis of the rRNA (see Supplementary Fig. 15b) 28 .…”

“…To provide further examples that show the potential to describe maturation and processing events for prokaryotic rRNA, we sequenced an H. volcanii wildtype strain grown under low salt conditions (see Supplementary Fig. 15b,c) 28 . Under these conditions, a 16S rRNA precursor with an extended 5’UTR (position - 121 – 5’bulge) appears that is only enriched in this condition.…”

“…For P. furiosus gene abundances from ONT data were compared to fragmented RNA sequencing data of mixed growth conditions (conditions, library, sequencing, mapping described in 31 ), by applying a regularized log transformation as described earlier 43 . For H. volcanii comparison, raw reads of a mixed RNA sequencing were extracted from the SRA (SRR7811297) 28 trimmed using trimmomatic 51 , (leading:20, trailing:20, slidingwindow:4:20, minlen:12), mapped to the reference genome using bowtie2 (-N 0, -L 26) 52 , converted to sorted bam files using samtools 41 and compared to ONT data as described for P. furiosus . Illumina RNA sequencing data for E. coli were also extracted from the NCBI (SRP056485, 37°C LB), analysed as described for H. volcanii Illumina data and also compared to the ONT reference data.…”

Regulated Iron Siderophore Production of the Halophilic Archaeon Haloferax volcanii

“…In the fully induced state, small amounts of three smaller transcripts were visible, which might originate from partial transcription termination. However, notably, the major transcript is about 9000 nt in length and is much longer than an average transcript, which has a length of about 1000 nt [12]. One major large transcript of about 9000 nt was also observed with probes for the genes HVO_B0041, HVO_B0042, HVO_B0043, and HVO_B0046, underscoring co-transcription of the six genes HVO_B0046 to HVO_B0041 into one common hexacistronic transcript.…”

“…Recently, the transcriptome of H. volcanii was analyzed with dRNA-Seq to identify transcription start sites (TSS) [11] and with RNA-Seq to characterize transcript lengths and operon structure [12]. The results were visualized with the Integrated Genome Browser [13] to get a first impression of the transcripts of the HVO_B0041 to HVO_B0046 gene cluster.…”