铜绿假单胞菌中RsmY和RsmZ小非编码调控RNA的功能分析。,Journal of Bacteriology - X-MOL

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铜绿假单胞菌中RsmY和RsmZ小非编码调控RNA的功能分析。
Journal of Bacteriology ( IF 2.7 ) Pub Date : 2018-02-22 , DOI: 10.1128/jb.00736-17
Kayley H Janssen ₁ , Manisha R Diaz ₁ , Matthew Golden ₁ , Justin W Graham ₂ , Wes Sanders ₃ , Matthew C Wolfgang _{2,

3} , Timothy L Yahr ₄

Affiliation

铜绿假单胞菌是革兰氏阴性机会病原体，具有明显的急性和慢性毒力表型。急性毒力通常与III型分泌系统（T3SS）的表达有关，而慢性毒力则以生物膜形成为特征。与急性和慢性毒力有关的许多表型受RsmA和RsmF反向调控。RsmA和RsmF都是RNA结合蛋白CsrA家族的成员，并在转录后水平上调节蛋白的合成。RsmA活性由两个小的非编码调控RNA（RsmY和RsmZ）控制。生物信息学分析表明RsmY和RsmZ各自具有3或4个假定的RsmA结合位点。每个预测的结合位点包含在茎-环结构的环部分中呈现的GGA序列。RsmY和RsmZ调节RsmA，通过从靶标mRNA隔离这些蛋白质来获得RsmF。在这项研究中，我们使用了通过引物延伸和突变分析（SHAPE-MaP）化学分析的选择性2'-羟基酰化反应，以确定RsmY和RsmZ的二级结构以及功能分析，以表征每个GGA位点对RsmY / RsmZ活性的贡献。我们的数据表明，RsmA在RsmY和RsmZ上具有两个优先结合位点，而RsmF在RsmY和RsmZ上具有一个优先结合位点。尽管RsmF和RsmA具有一个共有的共有位点，但RsmF的结合特性比RsmA的限制性更严格。要点CsrA同源物存在于许多细菌中。机会病原体铜绿假单胞菌使用RsmA和RsmF反向调节与急性和慢性毒力表型有关的因子。RsmA对RsmY和RsmZ的亲和力高于RsmF。这项研究的目的是通过使用RsmY和RsmZ调控小RNA（sRNA）作为模型来了解RsmA和RsmF的差异结合特性。对RsmY和RsmZ上预测的RsmA / RsmF结合位点的诱变揭示了在体内控制RsmA和RsmF活性所需的位点具有相似性，而RsmA的结合相对耐受结合位点突变，而RsmF对除其中两个以外的所有突变都敏感。进一步证明，体内和体外对RsmF结合活性的要求比对RsmA的要求更为严格。

"点击查看英文标题和摘要"

Functional Analyses of the RsmY and RsmZ Small Noncoding Regulatory RNAs in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen with distinct acute and chronic virulence phenotypes. Whereas acute virulence is typically associated with expression of a type III secretion system (T3SS), chronic virulence is characterized by biofilm formation. Many of the phenotypes associated with acute and chronic virulence are inversely regulated by RsmA and RsmF. RsmA and RsmF are both members of the CsrA family of RNA-binding proteins and regulate protein synthesis at the posttranscriptional level. RsmA activity is controlled by two small noncoding regulatory RNAs (RsmY and RsmZ). Bioinformatic analyses suggest that RsmY and RsmZ each have 3 or 4 putative RsmA binding sites. Each predicted binding site contains a GGA sequence presented in the loop portion of a stem-loop structure. RsmY and RsmZ regulate RsmA, and possibly RsmF, by sequestering these proteins from target mRNAs. In this study, we used selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) chemistry to determine the secondary structures of RsmY and RsmZ and functional assays to characterize the contribution of each GGA site to RsmY/RsmZ activity. Our data indicate that RsmA has two preferential binding sites on RsmY and RsmZ, while RsmF has one preferential binding site on RsmY and two sites on RsmZ. Despite RsmF and RsmA sharing a common consensus site, RsmF binding properties are more restrictive than those of RsmA.IMPORTANCE CsrA homologs are present in many bacteria. The opportunistic pathogen Pseudomonas aeruginosa uses RsmA and RsmF to inversely regulate factors associated with acute and chronic virulence phenotypes. RsmA has an affinity for RsmY and RsmZ higher than that of RsmF. The goal of this study was to understand the differential binding properties of RsmA and RsmF by using the RsmY and RsmZ regulatory small RNAs (sRNAs) as a model. Mutagenesis of the predicted RsmA/RsmF binding sites on RsmY and RsmZ revealed similarities in the sites required to control RsmA and RsmF activity in vivo Whereas binding by RsmA was relatively tolerant of binding site mutations, RsmF was sensitive to disruption to all but two of the sites, further demonstrating that the requirements for RsmF binding activity in vivo and in vitro are more stringent than those for RsmA.

更新日期：2019-11-01

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