The Transcriptome of the Human Pathogen Trypanosoma brucei at Single-Nucleotide Resolution

Abstract

The genome of Trypanosoma brucei, the causative agent of African trypanosomiasis, was published five years ago, yet identification of all genes and their transcripts remains to be accomplished. Annotation is challenged by the organization of genes transcribed by RNA polymerase II (Pol II) into long unidirectional gene clusters with no knowledge of how transcription is initiated. Here we report a single-nucleotide resolution genomic map of the T. brucei transcriptome, adding 1,114 new transcripts, including 103 non-coding RNAs, confirming and correcting many of the annotated features and revealing an extensive heterogeneity of 5′ and 3′ ends. Some of the new transcripts encode polypeptides that are either conserved in T. cruzi and Leishmania major or were previously detected in mass spectrometry analyses. High-throughput RNA sequencing (RNA-Seq) was sensitive enough to detect transcripts at putative Pol II transcription initiation sites. Our results, as well as recent data from the literature, indicate that transcription initiation is not solely restricted to regions at the beginning of gene clusters, but may occur at internal sites. We also provide evidence that transcription at all putative initiation sites in T. brucei is bidirectional, a recently recognized fundamental property of eukaryotic promoters. Our results have implications for gene expression patterns in other important human pathogens with similar genome organization ( Trypanosoma cruzi, Leishmania sp.) and revealed heterogeneity in pre-mRNA processing that could potentially contribute to the survival and success of the parasite population in the insect vector and the mammalian host.

Author Summary

Identifying genes essential for survival in the host is fundamental to unraveling the biology of human pathogens and understanding mechanisms of pathogenesis. The protozoan parasite Trypanosoma brucei causes devastating diseases in humans and animals in sub-Saharan Africa, and the publication in 2005 of the genome sequence provided the first glance at the coding potential of this organism. Although at present there is a catalogue of predicted protein coding genes, the challenge remains to identify all authentic genes, including their boundaries. We used next generation RNA sequencing (RNA-Seq) to map transcribed regions and RNA polymerase II transcription initiation sites on a genome-wide scale. This approach allowed us to improve and correct the current annotation, to reveal a widespread heterogeneity of RNA processing sites ( trans-splicing and polyadenylation) and to estimate that most genes are expressed at levels corresponding to 1 to 10 mRNAs per cell. Our data indicate that different transcript forms representing the same gene are present stochastically within the mRNA population. This unanticipated scenario may contribute to determining gene expression landscapes to adapt to different environments in the parasite life cycle.