Nanopore-based direct RNA sequencing of the Trypanosoma brucei transcriptome identifies novel lncRNAs

Abstract

Trypanosomatids are single-cell eukaryotic parasites. Unlike higher eukaryotes, they control gene expression posttranscriptionally and not at the level of transcription initiation. This involves all known cellular RNA circuits, from mRNA processing to mRNA decay to translation, in addition to a large panel of RNA-interacting proteins that modulate mRNA abundance. However, other forms of gene regulation, for example, by lncRNAs, cannot be excluded. LncRNAs are poorly studied in trypanosomatids, with only a single lncRNA characterized today. Furthermore, it is not clear whether the complete inventory of trypanosomatid lncRNAs is known because of the inherent cDNA recoding and DNA amplification limitations of short-read RNA sequencing. Here we overcome these limitations by using long-read direct RNA sequencing (DRS) on nanopore arrays. We analyze the native RNA pool of the two main lifecycle stages of the African trypanosome T. brucei with a special emphasis on the inventory of lncRNAs. We identify 207 previously unknown lncRNAs, 109 of which are stage-specifically expressed. We also present insights into the complexity of the T. brucei transcriptome, including alternative transcriptional start and stop sites and potential transcript isoforms to provide a bias-free understanding of the intricate RNA landscape in T. brucei.