Genome-wide analysis of mRNA abundance in two life-cycle stages of Trypanosoma brucei and identification of splicing and polyadenylation sites

Abstract

Transcription of protein-coding genes in trypanosomes is polycistronic and gene expression is primarily regulated by post-transcriptional mechanisms. Sequence motifs in the untranslated regions regulate mRNA trans-splicing and RNA stability, yet where UTRs begin and end is known for very few genes. We used high-throughput RNA-sequencing to determine the genome-wide steady-state mRNA levels (‘transcriptomes’) for ∼90% of the genome in two stages of the Trypanosoma brucei life cycle cultured in vitro. Almost 6% of genes were differentially expressed between the two life-cycle stages. We identified 5′ splice-acceptor sites (SAS) and polyadenylation sites (PAS) for 6959 and 5948 genes, respectively. Most genes have between one and three alternative SAS, but PAS are more dispersed. For 488 genes, SAS were identified downstream of the originally assigned initiator ATG, so a subsequent in-frame ATG presumably designates the start of the true coding sequence. In some cases, alternative SAS would give rise to mRNAs encoding proteins with different N-terminal sequences. We could identify the introns in two genes known to contain them, but found no additional genes with introns. Our study demonstrates the usefulness of the RNA-seq technology to study the transcriptional landscape of an organism whose genome has not been fully annotated.