作用于cip-cel mRNA的核糖核酸内切酶的鉴定

Identification of Ruminiclostridium cellulolyticum endoribonuclease specific to cip-cel mRNA

【目的】本研究以革兰氏阳性细菌解纤维素梭菌(Ruminiclostridium cellulolyticum)为研究对象,筛选作用于纤维小体编码基因簇cip-cel mRNA的核糖核酸内切酶。【方法】通过对预测的4个假定编码核糖核酸内切酶基因进行基因敲除、体内过表达、体外过表达和活性分析等方法,分析它们对cip-cel mRNA剪切位点的剪切能力。【结果】敲除rnc和rnj基因,对cip-cel mRNA剪切没有任何影响;体内过表达RNase时能够加速cip-cel mRNA的降解,而过表达RNase G时,则结果与野生型对照菌株无异;RNase G基因rng和RNase Y基因rny的体外活性鉴定分析,发现RNase G对体外转录的包含cip-cel mRNA剪切位点的RNA没有作用,而RNase Y能够对其进行剪切和降解。【结论】RNase Y是能够作用于cip-cel mRNA的核酸内切酶。该研究结果对理解革兰氏阳性细菌核糖核酸内切酶的作用机制,及其在转录后水平的调控基因差异表达等方面具有重大意义。

[Objective] In this study, the endoribonuclease processing the cip-cel mRNA encoding cellulosome was identified in Ruminiclostridium cellulolyticum. [Methods] The activity of four putative endoribonucleases(RNase Ⅲ, RNase J, RNase G, and RNase Y) in cip-cel mRNA cleavage was analyzed via gene knockout by Clostron, overexpression in vivo, overexpression in vitro, and activity analysis. [Results] Genes(rnc and rnj) encoding RNase Ⅲ and RNase J were disrupted and the resulted mutants did not affect the processing in intergenic region(IR) of the cip-cel mRNA. Moreover,RNase G and RNase Y were overexpressed and purified in vitro. RNase Y could cleave and degrade the mRNA harboring IR of the cip-cel mRNA in vitro, while RNase G failed to have any effect on that.Furthermore, overexpression of RNase Y in vivo could accelerate the degradation of cip-cel mRNA.[Conclusion] The cip-cel mRNA is potentially processed by RNase Y. The result helps deepen the understanding of the function of RNase Y in Gram-positive bacteria and the enzyme’s regulation of differential gene expression at the post-transcription level.