Extensive stage-regulation of translation revealed by ribosome profiling of Trypanosoma brucei

Abstract

BackgroundTrypanosoma 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.ResultsWe used ribosome profiling to examine genome-wide protein synthesis 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 appeared to be controlled by this 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 revealed stage-specific changes in gene expression that transcend strain and growth conditions. Genes with upstream ORFs had lower mean translation efficiency, but no evidence was found for involvement of uORFs in stage-regulation.ConclusionsRibosome profiling revealed that differences in the production of specific proteins in T. brucei bloodstream and procyclic forms are more extensive than predicted by analysis of mRNA abundance. While in vivo and in vitro derived bloodstream forms from different strains are more similar to one another than to procyclic forms, they showed many differences at both the mRNA and protein production level.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-911) contains supplementary material, which is available to authorized users.

The use of duplex-specific nuclease in ribosome profiling and a user-friendly software package for Ribo-seq data analysis

“…Profiling has proven to be increasingly valuable in studies of the translation process, for example, in the discovery of novel open reading frames (ORFs), the determination of elongation rates, the identification of sites of ribosome pausing and in the study of protein folding (for review, see Morris 2009;Weiss and Atkins 2011;Michel and Baranov 2013;Ingolia 2014;Jackson and Standart 2015). It also has broad application in the analysis of global gene expression and has been exploited in studies of infectious diseases (Stern-Ginossar et al 2012, 2015Liu et al 2013;Arias et al 2014;Caro et al 2014;Jensen et al 2014;Muzzey et al 2014;Vasquez et al 2014;Yang et al 2015), cell growth, differentiation and development Huang et al 2013;Lee et al 2013;Stadler and Fire, 2013;Stumpf et al 2013;Subramaniam et al 2013;Baudin-Baillieu et al 2014;Brubaker et al 2014;Duncan and Mata 2014;Gonzalez et al 2014;Hendriks et al 2014;Katz et al 2014;Kronja et al 2014;Schrader et al 2014;Vaidyanathan et al 2014;de Klerk et al 2015), apoptosis (Wiita et al 2013), mitochondrial gene expression and disease (Rooijers et al 2013;Williams et al 2014), cell stress (Gerashenko et al 2012;Labunskyy et al 2014;Zid and O'Shea 2014;Sidrauski et al 2015), cell toxicity …”

Bloodstream form Trypanosoma brucei do not require mRPN1 for gRNA processing

“…The reported low level of mRPN1 translation appears insufficient for an enzyme responsible for processing gRNAs, which are essential. Additional ribosome profiling data with more extensive and deeper sequencing depth similarly show mRPN1 is in the bottom 16th percentile (procyclic) or bottom 10th percentile (bloodstream) among ∼9100 transcripts ranked; GRBC1/GRBC2 were again between 88th and 92nd percentile in both life cycle stages, while KRET1 was found in the 71st and 53rd percentiles in procyclic and bloodstream cells, respectively (Jensen et al 2014). By way of comparison, ribosomeprotected mRNAs above the bottom 16th percentile in this procyclic stage data include VSG pseudogenes (e.g., Tb927.5.4900, Tb927.9.17390, Tb927.10.16470) and JBP1 (Tb927.11.13640), proteins that are expected to have no function in this life cycle stage and are considered to be "silent" (Van Leeuwen et al 1998;Pays 2005).…”

Illuminating Parasite Protein Production by Ribosome Profiling

“…While ribosome profiling has been utilized in a number of model organisms and human cells (for example, [7–9]), the approach has only recently seen application in infectious disease biology. Key examples include probing the effects of viral infection on host cells [10], investigating the mechanism of macrolide inhibition [11], and describing developmental regulation of protein production in the protozoan parasites Trypanosoma brucei [12,13], Trypanosoma cruzi [14], and Plasmodium falciparum [15]. Additionally it has been employed in gene discovery in T. brucei [16] and viruses [3,17].…”

“…Thus far it has been systematically applied to genome curation only for T. brucei [16]. There, we consolidated data from two earlier ribosome profiling studies [12,13] along with other types of data [24–27]. [24]Data visualization using the genome browser and annotation tool Artemis [28] allowed us to eliminate over six hundred T. brucei CDSs where the ribosome profiling, mRNA and/or SL RNA-seq evidence data contradicted the gene model.…”

“…The ribosome profiling data suggested that, in trypanosomatids, the CDS start codon is almost always the first ATG downstream of the major SL site, although some obvious upstream open reading frames (uORFs) were observed [12,13]. Mapping of the SL attachment sites suggested that many annotated start codons required revision [25–27].…”