Nanopore sequencing of RNA and cDNA molecules expands the transcriptomic toolbox in prokaryotes

Similar to its bacterial and eukaryotic counterparts, ribosomal RNA maturation in archaea is a multi-step process requiring well-defined endo- and exoribonuclease activities. However, the detailed rRNA processing pathway in archaea remained elusive. Here, we employed long-read direct cDNA and direct RNA Nanopore-based sequencing to study rRNA maturation in three archaeal model organisms, namely the Euryarchaea Haloferax volcanii and Pyrococcus furiosus and the Crenarchaeon Sulfolobus acidocaldarius. Compared to standard short-read protocols, nanopore sequencing facilitates simultaneous readout of 5- and 3-positions, which is required for the classification of rRNA processing intermediates. More specifically, we i) accurately detect and describe rRNA maturation stages by analysis of terminal read positions of cDNA reads and thereupon ii) explore the stage-dependent installation of the KsgA-mediated dimethylations in Haloferax volcanii using basecalling and signal characteristics of direct RNA reads. Due to the single-molecule sequencing capacity of nanopore sequencing, we could detect hitherto unknown intermediates with high confidence revealing details about the maturation of archaea-specific circular rRNA intermediates. Taken together, our study delineates common principles and unique features of rRNA processing in euryarchaeal and crenarchaeal representatives, thereby providing a comprehensive picture of rRNA maturation pathways in archaea.