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

Date Published: April 30, 2019

Publisher: Public Library of Science

Author(s): Sebastian Laass, Vivian A. Monzon, Jana Kliemt, Matthias Hammelmann, Friedhelm Pfeiffer, Konrad U. Förstner, Jörg Soppa, Akio Kanai.


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.

Partial Text

Small non-coding RNAs (sRNAs) exist in all three domains of life, archaea, bacteria, and eukaryotes. They were found to fulfill important regulatory roles and are involved in many biological functions, e.g. stress adaptation, metabolic regulation, and pathogenesis. In eukaryotes, altered expression levels of so called microRNAs (miRNAs), very small RNAs of about 22 nt, are associated with diseases. Several recent reviews summarize the current knowledge about sRNAs in eukaryotes [1–3], in bacteria [4–11], and in archaea [11–14].

The mixed RNA-Seq results in comparison with previous dRNA-Seq results and with the genome annotation yielded an unprecedented overview of the H. volcanii transcriptome, e.g. length distributions of all four RNA classes, operon analysis, and approximate 5’-/3’-UTR lengths. Northern blot analyses could exemplify characteristic features of the transcriptome, e.g. complex transcript patterns from gene clusters, differential formation of RNA isoforms, the existence of very long UTRs, and a high fraction of overlapping 3’-ends of cdRNAs. These results are a good starting point for further analyses, aiming to more deeply unravel RNA-based regulation in haloachaea.




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