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

Felix Grünberger; Robert Knüppel; Michael Jüttner; Martin Fenk; Andreas Borst; Robert Reichelt; Winfried Hausner; Jörg Soppa; Sebastien Ferreira-Cerca; Dina Grohmann

In this study, we have assessed the suitability of native RNA sequencing to obtain information on 696 the rRNA maturation pathway of different prokaryotes. Ribosomal RNA maturation proceeds via 697 the coordinated and defined order of ribonucleases action (exonucleolytic and/or endonucleolytic 698 cleavages) which generate pre-rRNA intermediates with defined premature rRNA 699 sequences 66,74,98,99 . The establishment of faithfull rRNA maturation maps in model organisms, like 700 E. coli, S. cerevisiae or human cell culture has required numerous analyses over the past decades 701 66,74,98,99 , and remains a technical challenge. Therefore, methodologies that might accelerate the 702 systematic analysis of rRNA maturation pathways across the tree of life, thereby enabling to 703 unravel the diversity of rRNA maturation strategies need to be established. Beyond the 704 identification of processing sites, the order of the processing events which can be, in part, deduced 705 from co-occurrence analysis of the 5´and 3´extremities is of biological relevance 66,74,98,99 . Whereas 706 we could confirm and extend our general view on the rRNA maturation pathway in archaea, the 707 3´-5´ processivity of Nanopore native RNA sequencing observed for rRNA impedes the accurate 708 analysis of pre-rRNA extremities co-segregation (see Fig. 5 and Supplementary Fig. 9b) . Supplementary Fig. 6) . c, Length of 3´ UTRs is based on the prediction 825 of transcription termination sites (TTS) and the comparison to annotated gene ends. Distribution of lengths is shown 826 for Nanopore data sets (purple) and compared to a Term-Seq E. coli Illumina data set (light-green) 108 . d, MEME 827 analysis 45 of extracted sequences upstream of Nanopore-predicted TSS reveals bacterial (position -10) and archaeal- The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2019.12.18.880849 doi: bioRxiv preprint