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Advancing Trypanosoma brucei genome annotation through ribosome profiling and spliced leader mapping

ABSTRACT: Since the initial publication of the trypanosomatid genomes, curation has been ongoing. Here we apply the technique of ribosome profiling to Trypanosoma brucei, identifying 223 new coding regions by virtue of ribosome occupancy in the corresponding transcripts. A small number of these putative genes correspond to extra copies of previously annotated genes but 85% are novel. The median size of these novels CDSs is small (74 aa) indicating that past annotation work has excelled at detecting large CDSs. Of the unique CDSs discovered here, over half have candidate orthologues in other trypanosomatid genomes, most of which were not yet annotated as genes. Still, approximately one-third of the new CDSs were found only in T. brucei subspecies. When combined with RNA-seq and spliced leader mapping, we were able to definitively revise the start sites for 430 CDSs as compared to the current gene models. Such data also allowed us to use a structured approach to eliminate 701 putative genes as protein-coding. Finally, the data pointed to several regions of the genome that had sequence errors that altered coding region boundaries.

ORGANISM(S): Trypanosoma brucei

PROVIDER: GSE72463 | GEO | 2015/12/22

SECONDARY ACCESSION(S): PRJNA294100

REPOSITORIES: GEO

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Project description:Trypanosoma brucei subspecies infect humans and animals in sub-Saharan Africa. This early diverging eukaryote shows many novel features in basic biological processes, including the use of polycistronic transcription to generate all protein-coding mRNAs. Therefore we hypothesized that translational control provides a means to tune gene expression during parasite development in mammalian and fly hosts. We used ribosome profiling to examine genome-wide protein production in animal-derived slender bloodstream forms and cultured procyclic (insect midgut) forms. About one-third of all CDSs showed statistically significant regulation of protein production between the two stages. Of these, more than two-thirds showed a change in translation efficiency, but few were controlled by this parameter alone. Ribosomal proteins were translated poorly, especially in animal-derived parasites. A disproportionate number of metabolic enzymes were up-regulated at the mRNA level in procyclic forms, as were variant surface glycoproteins in bloodstream forms. Comparison with cultured bloodstream forms from another strain identified stage-specific changes in protein production that transcend strain and growth conditions. Genes with upstream ORFs had a lower mean translation efficiency but no evidence was seen for involvement of these ORFs in stage-regulation. Ribosome profiling revealed that differences in the production of specific proteins in T. brucei slender bloodstream and procyclic forms are more common than anticipated from analysis of mRNA abundance. While in vivo and in vitro derived slender bloodstream forms of different strains are more similar to one another than to procyclic forms, they showed numerous differences at both the mRNA and protein production level. Ribosome profiling and mRNA libraries were constructed in triplicate from in vitro PCF and in vivo BF lifestages of theT. brucei Treu927 and in vitro T. brucei Lister427, to evaluate role of translational gene regulation

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Advancing Trypanosoma brucei genome annotation through ribosome profiling and spliced leader mapping

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