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

在以前的工作中，我们在玛氏甲烷链霉菌中鉴定了248个候选调控RNA，包括mRNA5‘或3’区的反义sRNA(AsRNAs)，以及含有≤30个氨基酸的小ORF(SpRNAs)的sRNAs。甲烷肉毒目的高度保守性和对氮(N)反应的大量差异转录的sRNAs证明了sRNAs在M.maazei对N胁迫反应中的重要作用。我们现在的目标是：(I)研究一些可能参与N调节的已验证的sRNAs的调节作用和作用模式(例如sRNA154)，(Ii)阐明AmtR作为潜在的RNA结合蛋白的作用并验证其在N胁迫反应中的拟议作用，(Iii)鉴定sRNAs中发现的小ORF，并阐明各自作为mRNA和sRNAs的转录本的潜在双重功能，以及(Iv)通过转录后asRNA介导的转座酶表达控制来研究转座事件与N可利用性的关系。为了实现这些主要目标，将为玛泽分枝杆菌建立几种新的方法，包括四环素诱导的启动子用于sRNA的脉冲表达，以及将各种RNA标签与sRNA融合以识别相互作用的伙伴。总体而言，拟议的项目将允许解开sRNA在M.mazei的N-调节子中的作用，并对拟议的转位事件做出更深入的了解，以响应M.maazei的N有效性。

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.