sRNA₄₁ affects ribosome binding sites within polycistronic mRNAs in Methanosarcina mazei Gö1

Abstract

Several noncoding RNAs potentially involved in nitrogen (N)-regulation have been detected in Methanosarcina mazei, however, targets have been identified only for one of them. Here, we report on the function of sRNA , highly expressed under N-sufficiency. Comprising 120 nucleotides, sRNA shows high sequence and structural conservation within draft genomes of numerous Methanosarcina species. In silico target prediction revealed several potential targets, including genes of two homologous operons encoding for acetyl-CoA-decarbonylase/synthase complexes (ACDS) representing highly probable target candidates. A highly conserved single stranded region of sRNA was predicted to mask six independent ribosome binding sites of these two polycistronic mRNAs and was verified in vitro by microscale thermophoresis. Proteome analysis of the respective sRNA -deletion mutant showed increased protein expression of both ACDS complexes in the absence of sRNA , whereas no effect on transcript levels was detected, arguing for sRNA -mediated post-transcriptional fine-tuning of ACDS expression. We hypothesize that the physiological advantage of downregulating sRNA under N-limiting conditions is the resulting increase of ACDS protein levels. This provides sufficient amounts of amino acids for nitrogenase synthesis as well as reducing equivalents and energy for N -fixation, thus linking the carbon and N-metabolism.

The Non-Coding Regulatory RNA Revolution in Archaea

“…In contrast, they also showed that loop 2 of sRNA154 contains anti-ribosome binding site (RBS) sequences that masked the RBS of the glnA2-mRNA target, repressing translation initiation [2]. Another sRNA in M. mazei, sRNA41, was also found to repressed its targets at the translation level by masking ribosome binding sites within polycistronic mRNAs [13]. Other molecular mechanisms have been identified in the archaea such as the binding of itsRNAs to the 3'UTR of mRNA targets in S. solfataricus [34] and, more recently, in H. volcanii (Kliemt, Jaschinski, and Soppa, unpublished data).…”

“…Most recently, another M. mazei itsRNA, sRNA41, was found to be down-regulated during nitrogen limiting conditions. Targets of sRNA41 were involved in acetyl-CoA-decarbonylase/synthase complexes (ACDS) and were repressed at the translational level [13]. Thus sRNA41 was predicted to play a role in repressing ACDS protein levels, however, under nitrogen limiting conditions, sRNA41 was down-regulated, thus allowing ACDS levels to increase, which in turn provided sufficient amino acids for nitrogenase synthesis and energy for N2-fixation [13].…”

“…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.…”

“…Targets of sRNA41 were involved in acetyl-CoA-decarbonylase/synthase complexes (ACDS) and were repressed at the translational level [13]. Thus sRNA41 was predicted to play a role in repressing ACDS protein levels, however, under nitrogen limiting conditions, sRNA41 was down-regulated, thus allowing ACDS levels to increase, which in turn provided sufficient amino acids for nitrogenase synthesis and energy for N2-fixation [13]. While these studies provide great examples of gene network regulated by sRNAs in the archaea, additional work is needed to identify many more molecular targets of archaeal itsRNAs and the diverse mechanisms of their sRNA-mRNA interactions.…”

“…sRNAs in bacteria and eukarya play essential roles in transcriptional regulation, chromosome replication, RNA processing and modification, mRNA stability and translation, and even protein degradation and translocation [3][4][5]. Recently, it was discovered that archaeal genomes encode for large numbers of sRNAs and that many of them are responsive to environmental stresses [1, 2,[6][7][8][9][10][11][12][13][14].…”