十字花科黑腐病菌非编码RNA SR220与RsmA的相互作用关系及SR220剪切位点的初步鉴定

The Interaction between the Non-coding RNA SR220 and RsmA and the Preliminary Identification of the SR220 Cleavage Site in Xanthomonas campestris pv. campestris

次生代谢抑制(Repression of secondary metabolism,Rsm)调控系统是一个由RNA结合蛋白RsmA以及非编码RNA组成的广泛存在于细菌中的全局转录后调控系统。RsmA蛋白是Rsm调控系统的核心,它通过与靶标mRNA结合来控制基因表达;而非编码RNA的作用是抑制RsmA蛋白的调控活性。研究表明,在一些细菌中,RsmA可以通过复杂的调控网络在转录水平正向调控这些抑制它的非编码RNA的表达。本实验室前期研究发现,在十字花科黑腐病菌(Xanthomonas campestris pv.campestris,Xcc)中,SR220是抑制RsmA活性的非编码RNA,但RsmA是否调控SR220的表达目前还不清楚。本研究使用Northern杂交技术检测和比较了野生型菌株和rsmA缺失突变体中SR220的表达水平。结果发现,尽管在野生型菌株中SR220正常表达,但在rsmA缺失突变体中却检测不到SR220的信号,证明SR220的产生需要RsmA。更重要的是,进一步的实验发现,SR220的产生需要其与RsmA直接结合,说明RsmA是在转录后水平,而不是像其他细菌在转录水平调控抑制其非编码RNA(SR220)的合成。本实验室之前已经证明,SR220是由XC1332 mRNA 3′非翻译区剪切加工而来。本研究经鉴定发现,在XC1332的终止密码子TAA到SR220的第一个核苷酸之间的25 bp间隔区存在1个关键的剪切位点。

The Rsm(Repression of secondary metabolism)regulatory system,consisting of the RNA-binding protein RsmA and a few non-coding RNAs,is a global post-transcriptional regulatory system widely present in bacteria.The RsmA protein is the core component of the Rsm regulatory system,it controls gene expression by binding to its target mRNAs.The role of the Rsm non-coding RNAs is to inhibit the regulatory activity of RsmA.It has been shown in some bacteria species that RsmA activates the expression of the RsmA-inhibiting non-coding RNAs at the transcriptional level via a complicated regulatory network.Our laboratory previously found that the non-coding RNA SR220 can binds to and then inhibits the activity of RsmA in Xanthomonas campestris pv.campestris(Xcc).However,whether RsmA regulates the expression of SR220 is still unclear so far.In this study,the expression level of SR220 in the wild-type strain and the rsmA deletion mutant were detected and compared by Northern hybridization analysis.The results showed that SR220 can only be detected in the wild-type strain but not the rsmA deletion mutant,demonstrating that RsmA is required for the biosynthesis of SR220.More importantly,further experiments revealed that binding with RsmA directly is essential for SR220 biosynthesis,indicating that RsmA regulates the RsmA-inhibiting non-coding RNA SR220 at the post-transcriptional level,diffe-rent from the situation in other bacteria that RsmA regulates the expression of the RsmA-inhibiting non-coding RNAs at the transcriptional level.Our laboratory has previously demonstrated that SR220 is spliced from the 3′untranslated region of XC1332 mRNA.Preliminary identification found that there is a key cleavage site in the 25 bp spacer between the termination codon TAA of XC1332 and the first nucleotide of SR220.