Nanopore-based RNA sequencing deciphers the formation, processing, and modification steps of rRNA intermediates in archaea

Abstract

Ribosomal RNA (rRNA) maturation in archaea is a complex multi-step process that requires well-defined endo- and exoribonuclease activities to generate fully mature linear rRNAs. However, technical challenges prevented detailed mapping of rRNA processing steps and a systematic analysis of rRNA maturation pathways across the tree of life. In this study, we employed long-read (PCR)-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 significantly expanding our understanding of rRNA maturation pathways in archaea.

Design and Construction of a Multi-Tiered Minimal Actin Cortex for Structural Support in Lipid Bilayer Applications

“…A number of emerging sensing technologies critically depend on robust lipid bilayers, including those based on biological nanopores. − To work properly, analytes must be able to move from the bathing solution surrounding the interface to the mouth of the nanopore. Specific nanopore sensing applications include DNA sequencing, RNA sequencing, − nucleic acid detection, − polypeptide detection, RNA profiling, synthetic polymer characterization, − digital data storage, − disease detection, ion sensing, small molecule detection, and sensing of protein–drug interactions . Multiple types of nanopores with various pore sizes and channel structures can be employed.…”