Rho在Rho依赖性终止子处促进Rho依赖性终止(RDT),在mRNA的3'端产生一个没有二级结构的可变长度区。确定体内确切的RDT位点具有挑战性,因为RDT后通过3'至5'核酸外切酶处理迅速去除mRNA的3'末端。这里,我们应用合成小RNA(sysRNA)通过利用其互补碱基配对靶向mRNA的能力,在体内鉴定RDT区.通过对cDNA3'末端快速扩增的联合分析,引物延伸,和毛细管电泳,我们可以精确地绘制和定量mRNA3'末端。我们发现在sysRNA和mRNA之间形成的互补双链RNA(dsRNA)在dsRNA区域的中间被RNA酶III有效地切割。dsRNA的形成似乎保护切割的RNA3'末端免受3'至5'核酸外切酶的快速降解,从而稳定mRNA3'端。我们进一步验证了3'端的信号强度与mRNA的量呈正相关。通过构建一系列具有紧密靶位点的sysRNAs,并比较野生型和Rho受损菌株3'末端的信号强度差异,我们最终确定了一个在21bp范围内mRNA表达增加的区域,被确定为RDT区域。我们的结果证明了使用sysRNA作为一种新工具来识别体内RDT区域并扩展sysRNA的应用范围的能力。重要性sysRNA,以前被广泛使用,由于抑制效果不稳定和抑制效率低等问题,随着更多抑制基因表达的新技术的出现,已经稳步失去了普及。然而,由于其易于设计和对细胞的低代谢负担,它仍然是一个有趣的话题。这里,第一次,我们发现了一种使用sysRNA在体内鉴定RDT区域的新方法。这个新特征很重要,因为自1969年发现Rho蛋白以来,由于核酸外切酶对RNA3'末端的快速加工,体内RDT位点的特异性鉴定一直很困难。和sysRNA可能提供一种新的方法来应对这一挑战。
Rho promotes Rho-dependent termination (RDT) at the Rho-dependent terminator, producing a variable-length region without secondary structure at the 3\' end of mRNA. Determining the exact RDT site in vivo is challenging, because the 3\' end of mRNA is rapidly removed after RDT by 3\'-to-5\' exonuclease processing. Here, we applied synthetic small RNA (sysRNA) to identify the RDT region in vivo by exploiting its complementary base-pairing ability to target mRNA. Through the combined analyses of rapid amplification of cDNA 3\' ends, primer extension, and capillary electrophoresis, we could precisely map and quantify mRNA 3\' ends. We found that complementary double-stranded RNA (dsRNA) formed between sysRNA and mRNA was efficiently cleaved by RNase III in the middle of the dsRNA region. The formation of dsRNA appeared to protect the cleaved RNA 3\' ends from rapid degradation by 3\'-to-5\' exonuclease, thereby stabilizing the mRNA 3\' end. We further verified that the signal intensity at the 3\' end was positively correlated with the amount of mRNA. By constructing a series of sysRNAs with close target sites and comparing the difference in signal intensity at the 3\' end of wild-type and Rho-impaired strains, we finally identified a region of increased mRNA expression within the 21-bp range, which was determined as the RDT region. Our results demonstrated the ability to use sysRNA as a novel tool to identify RDT regions in vivo and expand the range of applications of sysRNA. IMPORTANCE sysRNA, which was formerly widely employed, has steadily lost popularity as more novel techniques for suppressing gene expression come into existence because of issues such as unstable inhibition effect and low inhibition efficiency. However, it remains an interesting topic as a regulatory tool due to its ease of design and low metabolic burden on cells. Here, for the first time, we discovered a new method to identify RDT regions in vivo using sysRNA. This new feature is important because since the discovery of the Rho protein in 1969, specific identification of RDT sites in vivo has been difficult due to the rapid processing of RNA 3\' ends by exonucleases, and sysRNA might provide a new approach to address this challenge.