rRNA processing in Archaea using Nanopore-based RNA-seq

Similar to its bacterial and eukaryotic counterparts, ribosomal RNA maturation in archaea is a multi-step process, which requires the action of well-defined endo- and exoribonuclease activities. During this process, diverse enzyme or RNA-guided modifications are installed mainly at the functional centre of active ribosomes. However, only little is known about the detailed rRNA processing pathway in archaea. Here, we employed long-read direct cDNA and direct RNA Nanopore-based sequencing to study rRNA maturation and modification 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 rRNA stage classification. 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 dimethylation in Haloferax volcanii using base calling and signal characteristics of direct RNA reads. Due to the single-molecule sequencing capacity of Nanopore sequencing, we could detect extremely short-lived and hitherto unknown intermediates with high confidence revealing details about the formation of archaea-specific circular rRNA intermediates. Taken together, our study delineates common principles and unique features for rRNA processing in euryarchaeal and crenarchaeal representatives, thereby providing a comprehensive picture of rRNA maturation pathways in archaea.