Nanopore-based native RNA sequencing provides insights into prokaryotic transcription, operon structures, rRNA maturation and modifications

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 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 accurately (re-)annotate large transcriptional units and boundaries. Our analysis of transcription termination sites revealed that diverse termination mechanisms are in place in archaea. Moreover, we shed light on the poorly understood rRNA processing pathway in archaea and detected new processing intermediates. 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 rRNA methylation sites. Notably, our analysis suggests that rRNA modifications are more abundant in a hyperthermophilic organism.