Nanopore-Based Direct RNA Sequencing of the Trypanosoma brucei Transcriptome Identifies Novel lncRNAs

Trypanosomatids are single-cell eukaryotic parasites. Unlike higher eukaryotes, they control gene expression post-transcriptionally 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 to date. 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 Trypanosoma brucei, with a special emphasis on the inventory of lncRNAs. We identify 207 previously unknown lncRNAs, 32 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.