Marincola Gabriella, Wolz Christiane
Interfaculty Institute of Microbiology and Infection Medicine, University of Tübingen, Tübingen 72076, Germany.
Nucleic Acids Res. 2017 Jun 2;45(10):5980-5994. doi: 10.1093/nar/gkx296.
In gram-positive bacteria, RNase J1, RNase J2 and RNase Y are thought to be major contributors to mRNA degradation and maturation. In Staphylococcus aureus, RNase Y activity is restricted to regulating the mRNA decay of only certain transcripts. Here the saePQRS operon was used as a model to analyze RNase Y specificity in living cells. A RNase Y cleavage site is located in an intergenic region between saeP and saeQ. This cleavage resulted in rapid degradation of the upstream fragment and stabilization of the downstream fragment. Thereby, the expression ratio of the different components of the operon was shifted towards saeRS, emphasizing the regulatory role of RNase Y activity. To assess cleavage specificity different regions surrounding the sae CS were cloned upstream of truncated gfp, and processing was analyzed in vivo using probes up- and downstream of CS. RNase Y cleavage was not determined by the cleavage site sequence. Instead a 24-bp double-stranded recognition structure was identified that was required to initiate cleavage 6 nt upstream. The results indicate that RNase Y activity is determined by secondary structure recognition determinants, which guide cleavage from a distance.
在革兰氏阳性菌中，核糖核酸酶J1、核糖核酸酶J2和核糖核酸酶Y被认为是信使核糖核酸（mRNA）降解和成熟的主要贡献者。在金黄色葡萄球菌中，核糖核酸酶Y的活性仅限于调节某些转录本的mRNA衰变。在这里，saePQRS操纵子被用作模型来分析活细胞中核糖核酸酶Y的特异性。一个核糖核酸酶Y切割位点位于saeP和saeQ之间的基因间区域。这种切割导致上游片段的快速降解和下游片段的稳定。从而，操纵子不同组分的表达比例向saeRS偏移，强调了核糖核酸酶Y活性的调节作用。为了评估切割特异性，将sae切割位点（CS）周围的不同区域克隆到截短的绿色荧光蛋白（gfp）上游，并使用CS上下游的探针在体内分析加工过程。核糖核酸酶Y的切割不是由切割位点序列决定的。相反，鉴定出一个24碱基对的双链识别结构，该结构是在切割位点上游6个核苷酸处启动切割所必需的。结果表明，核糖核酸酶Y的活性由二级结构识别决定因素决定，这些因素在一定距离外引导切割。
