大腸桿菌中RprA sRNA對nagE與lamB mRNA之抑制調控機制

Abstract

非編碼的小核醣核酸(non-coding small RNA, sRNA)廣泛的存在於真核與原核生物中，並在許多生理途徑中扮演著調控者的角色。在大腸桿菌(Escherichia coli)中，已有將近一百多個sRNA被發現，其中有些sRNA與目標mRNA的配對需要RNA監護者(RNA chaperon) Hfq (host factor for phage Qβ)的幫助，sRNA的結合可以調節mRNA的穩定性或轉譯效率。 RprA為一個長105核苷酸(nucleotide)的sRNA，已知RprA sRNA利用鹼基配對(base pair)的方式與mRNA結合，且此過程需要Hfq的幫助，RprA sRNA會在生長停滯期(stationary phase)時表現，並且在受到高滲透壓的誘導時大量表現。目前已知文獻中指出，RprA sRNA於轉錄後階段調控目標，如rpoS和csgD mRNA，rpoS可轉譯為受環境壓力時所需要的sigma factor、csgD 為生物膜(biofilm)形成時所需的轉錄因子。根據實驗室先前的轉錄體學(transcriptome)研究中指出，過表現RprA sRNA降低了nagE和lamB mRNA的表現量，NagE為N-乙醯葡萄糖胺通透酶(N-acetylglucosamine permease)、LamB為噬菌體λ受體蛋白及麥芽糖外膜蛋白(phage lambda receptor protein/maltose outer membrane porin)。 藉由綠螢光融合報導系統(gfp reporter fusion system)，證實RprA sRNA於轉錄後階段負調控nagE和lamB mRNA，且RprA sRNA在Hfq的幫助下，主要藉由區域II負調控nagE::gfp的表現。RprA sRNA之區域II與nagE mRNA之5’-UTR結合的位置位於核醣體結合位(ribosome-binding site)，導致nagE mRNA的降解因而抑制了轉譯效率。然而對lamB的負調控則需要整段的RprA sRNA。且此兩目標的降解主要由RNase E來執行。最後，證實了nagE和lamB mRNA為RprA sRNA所負調控的新目標mRNA。

Non-coding small RNAs (sRNAs) which function as RNA regulators involve in various physiological pathways and widely distributes in pro- and eukaryotes. In Escherichia coli, approximate 100 sRNAs have been experimentally identified. Some of them interact with RNA chaperon, Hfq, and target to specific mRNAs by base pairing. The sRNA-binding can regulate mRNA stability or translation efficiency. The RprA, a 105 nt (nucleotide) sRNA, forms base pair with its target mRNAs by Hfq. RprA sRNA accumulates in the stationary phase and is dramatically induced upon hyperosmotic stress. It has been reported to regulate cognate targets in post-transcription level, such as rpoS and csgD encoding for general stress factor and transcription factor in biofilm formation, respectively. According to the comparative transcriptome in our previous study, overexpression of RprA sRNA decreased level of N-acetylglucosamine permease (NagE) and phage lambda receptor protein/maltose outer membrane porin (LamB). By using gfp reporter fusion, it showed that the RprA sRNA post-transcriptionally regulated nagE and lamB experssion. In addition, the region II in RprA sRNA was required for down-regulation of nagE::gfp expression, and this regulation was mediated by Hfq. The base-pairing of region II RprA sRNA to 5’UTR of nagE mRNA in the location of the ribosome-binding site was enhanced by the presence of Hfq, leading to nagE mRNA decay and subsequent translational inhibition. RprA sRNA was identified to be the whole sequence that negatively regulated lamB mRNA. The degradation of these two targets was done mainly by RNase E. Also, we demonstrated that the nagE and lamB mRNA, a novel target, was negatively regulated by RprA sRNA.