Functional analysis of selected sRNAs potentially involved in nitrogen and / or general stress response in the archaeon Methanosarcina mazei Gö1

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In previous work we identified 248 regulatory RNA (sRNA) candidates in Methanosarcina mazei, including sRNAs antisense to 5’ or 3’ regions of mRNAs (asRNAs), and sRNAs containing small ORFs of ≤ 30 amino acids (spRNAs). The strong conservation in Methanosarcinales and the high number of differentially transcribed sRNAs in response to nitrogen (N) argue for a prominent role of sRNAs in the N-stress response in M. mazei. We now aim to (i) study the regulatory role and mode of action of selected verified sRNAs potentially involved in N-regulation (e.g. sRNA154), (ii) elucidate the role of AmtR as a potential RNA binding protein and verify its proposed role in N-stress response, (iii) characterize the small ORFs identified in sRNAs and elucidate the potential dual function of the respective transcripts as mRNA and sRNAs, and (iv) study the proposed relationship of transposition events in M. mazei and N-availability by a post-transcriptional asRNA-mediated control of transposase expression. In order to achieve those major goals, several novel methods will be established for M. mazei including a tetracycline-inducible promoter for pulse-expression of sRNAs and fusing various RNA-tags to sRNAs to identify interacting partners. Overall, the proposed project will allow to unravel the role of sRNAs in the N-regulon of M. mazei and to achieve a deeper insight regarding the proposed transposition events in response to N availability in M. mazei.