Abstract
The haloarchaeon Haloferax volcanii contains nearly 2800 small non-coding RNAs (sRNAs). One intergenic sRNA, sRNA132, was chosen for a detailed characterization. A deletion mutant had a growth defect and thus underscored the importance of sRNA132. A microarray analysis identified the transcript of an operon for a phosphate-specific ABC transporter as a putative target of sRNA132. Both the sRNA132 and the operon transcript accumulated under low phosphate concentrations, indicating a positive regulatory role of sRNA132. A kinetic analysis revealed that sRNA132 is essential shortly after the onset of phosphate starvation, while other regulatory processes take over after several hours. Comparison of the transcriptomes of wild-type and the sRNA132 gene deletion mutant 30 min after the onset of phosphate starvation revealed that sRNA132 controls a regulon of about 40 genes. Remarkably, the regulon included a second operon for a phosphate-specific ABC transporter, which also depended on sRNA132 for rapid induction in the absence of phosphate. Competitive growth experiments of the wild-type and ABC transporter operon deletion mutants underscored the importance of both transporters for growth at low phosphate concentrations. Northern blot analyses of four additional members of the sRNA132 regulon verified that all four transcripts depended on sRNA132 for rapid regulation after the onset of phosphate starvation. Importantly, this is the first example for the transient importance of a sRNA for any archaeal and bacterial species. In addition, this study unraveled the first sRNA regulon for haloarchaea.
Highlights
Small non-coding regulatory RNAs have been found in all three domains of life
For reasons described above the sRNA132 was the first sRNA of H. volcanii that was chosen for a detailed characterization
Its 5 - and 3 -ends were determined by cRT-PCR and it was revealed that the sRNA132 has a length of 138 nt
Summary
Small non-coding regulatory RNAs (sRNAs) have been found in all three domains of life. SRNAs have been identified in all archaeal and bacterial species that were investigated. They are typically between 100 nt and 500 nt long and can be divided into three classes, Characterization of the sRNA132 Regulon of Haloferax volcanii i.e., intergenic sRNAs (igRNAs), cis antisense sRNAs (asRNAs), and cis sense RNAs (isRNAs). RNA-Seq and dRNA-Seq studies have been performed with a limited number or archaeal species, i.e., Haloferax volcanii (Babski et al, 2016; Gelsinger and DiRuggiero, 2018b; Laass et al, 2019), Methanolobus psychrophilus (Li et al, 2015), Methanosarcina mazei Gö1 (Jäger et al, 2009), Pyrococcus abyssi (ToffanoNioche et al, 2013), Sulfolobus solfataricus (Wurtzel et al, 2010), and Thermococcus kodakarensis (Jäger et al, 2014). The numbers were very different, e.g., only about 300 sRNAs were reported for S. solfataricus, while 2800 non-coding RNAs were found for H. volcanii
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