The prokaryotic transcriptome is shaped by transcriptional and posttranscriptional events that define the characteristics of an RNA, including transcript boundaries, the base modification status, and processing pathways to yield mature RNAs. Currently, a combination of several specialised short-read sequencing approaches and additional biochemical experiments are required to describe all transcriptomic features. In this study, we present native RNA sequencing of bacterial (E. coli) and archaeal (H. volcanii, P. furiosus) transcriptomes employing the Oxford Nanopore sequencing technology. Based on this approach, we could address multiple transcriptomic characteristics simultaneously with single-molecule resolution. Taking advantage of long RNA reads provided by the Nanopore platform, we could (re-)annotate large transcriptional units and boundaries. Our analysis of transcription termination sites suggests that diverse termination mechanisms are in place in archaea. Moreover, we shed additional light on the poorly understood rRNA processing pathway in Archaea. One of the key features of native RNA sequencing is that RNA modifications are retained. We could confirm this ability by analysing the well-known KsgA-dependent methylation sites and mapping of N4-acetylcytosines modifications in rRNAs. Notably, we were able to follow the relative timely order of the installation of these modifications in the rRNA processing pathway.

中文翻译：

---

使用基于纳米孔的天然RNA测序探索原核转录，操纵子结构，rRNA成熟和修饰

原核转录组受转录和转录后事件的影响，这些事件定义了RNA的特征，包括转录物边界，碱基修饰状态和产生成熟RNA的加工途径。目前，需要多种专门的短读测序方法和其他生化实验相结合来描述所有转录组特征。在这项研究中，我们介绍了采用牛津纳米孔测序技术的细菌（大肠杆菌）和古细菌（H. volcanii，P。furiosus）转录组的天然RNA测序。基于这种方法，我们可以用单分子分辨率同时解决多个转录组特征。利用Nanopore平台提供的长RNA读数，我们可以（重新）注释较大的转录单位和边界。我们对转录终止位点的分析表明，古细菌中存在多种终止机制。此外，我们进一步了解了古细菌中尚未充分了解的rRNA加工途径。天然RNA测序的关键特征之一是保留了RNA修饰。我们可以通过分析众所周知的KsgA依赖性甲基化位点并绘制rRNA中的N4-乙酰胞嘧啶修饰图来确认这种能力。值得注意的是，我们能够按照相对及时的顺序在rRNA加工途径中安装这些修饰。天然RNA测序的关键特征之一是保留了RNA修饰。我们可以通过分析众所周知的KsgA依赖性甲基化位点并绘制rRNA中的N4-乙酰胞嘧啶修饰图来确认这种能力。值得注意的是，我们能够按照相对及时的顺序在rRNA加工途径中安装这些修饰。天然RNA测序的关键特征之一是保留了RNA修饰。我们可以通过分析众所周知的KsgA依赖性甲基化位点并绘制rRNA中的N4-乙酰胞嘧啶修饰图来确认这种能力。值得注意的是，我们能够按照相对及时的顺序在rRNA加工途径中安装这些修饰。