An archaeal sRNA targeting cis - and trans -encoded mRNAs via two distinct domains

Abstract

We report on the characterization and target analysis of the small (s)RNA162 in the methanoarchaeon Methanosarcina mazei. Using a combination of genetic approaches, transcriptome analysis and computational predictions, the bicistronic MM2441-MM2440 mRNA encoding the transcription factor MM2441 and a protein of unknown function was identified as a potential target of this sRNA, which due to processing accumulates as three stabile 5′ fragments in late exponential growth. Mobility shift assays using various mutants verified that the non-structured single-stranded linker region of sRNA162 (SLR) base-pairs with the MM2440-MM2441 mRNA internally, thereby masking the predicted ribosome binding site of MM2441. This most likely leads to translational repression of the second cistron resulting in dis-coordinated operon expression. Analysis of mutant RNAs in vivo confirmed that the SLR of sRNA162 is crucial for target interactions. Furthermore, our results indicate that sRNA162-controlled MM2441 is involved in regulating the metabolic switch between the carbon sources methanol and methylamine. Moreover, biochemical studies demonstrated that the 5′ end of sRNA162 targets the 5′-untranslated region of the cis-encoded MM2442 mRNA. Overall, this first study of archaeal sRNA/mRNA-target interactions unraveled that sRNA162 acts as an antisense (as)RNA on cis- and trans-encoded mRNAs via two distinct domains, indicating that cis-encoded asRNAs can have larger target regulons than previously anticipated.

The Non-Coding Regulatory RNA Revolution in Archaea

“…While phenotypic characterization of sRNA deletion mutants, including 10 gain-of-function phenotypes out of 27 mutants tested, confirmed their roles in metabolic regulation, stress adaptation and complex behavior [12,36], their targets are still unknown with a few exceptions. Some of these exceptions comes from the study of M. mazei cultures grown under nitrogen starvation conditions where RNA-seq experiments revealed the differential expression of a number of sRNAs in response to nitrogen availability [23,37]. This then resulted in the identification of the first in vivo targets for archaeal intergenic sRNAs [23,37].…”

“…Some of these exceptions comes from the study of M. mazei cultures grown under nitrogen starvation conditions where RNA-seq experiments revealed the differential expression of a number of sRNAs in response to nitrogen availability [23,37]. This then resulted in the identification of the first in vivo targets for archaeal intergenic sRNAs [23,37]. A potential target for one of these sRNAs, sRNA162, was a bicistronic mRNA encoding for a transcription factor involved in regulating the switch between carbon sources and for a protein of unknown function [37].…”

“…This then resulted in the identification of the first in vivo targets for archaeal intergenic sRNAs [23,37]. A potential target for one of these sRNAs, sRNA162, was a bicistronic mRNA encoding for a transcription factor involved in regulating the switch between carbon sources and for a protein of unknown function [37]. Another sRNA, sRNA154, was also exclusively expressed during nitrogen starvation conditions and the multiple targets for sRNA154 included mRNAs for the α subunit of nitrogenase (nifH), the transcriptional activator of the nif operon (nrpA), and glutamine synthase1/2 (glnA1/glnA2).…”

“…To date, very few of the newly reported candidate sRNAs in the archaea have been functionally characterized [2,13,37] Co-immuno-precipitation with the Lsm protein, the archaeal homolog of Hfq, was used to "capture" sRNAs in vitro [6]. However, the role of Lsm -or any other RNA-binding proteinremains to be elucidated in the archaea.…”

Transcription Regulation in Archaea

“…The development and recent advances in genetic techniques for more archaeal species are now offering complementary in vivo studies to probe regulatory strategies and rationally manipulate protein interfaces and activities in the cell. Although discussion of transcriptome mapping of archaeal organisms is outside the scope of this review, the mapping is becoming more frequent (36,(184)(185)(186)(187)(188)(189) and offers invaluable insight into noncoding RNA, transcription start site selection and redundancy, and expression levels under various growth conditions (36,171,(190)(191)(192)(193).…”