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

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Abstract

Background

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.

Results

We 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.

Conclusions

Ribosome 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.

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The online version of this article (doi:10.1186/1471-2164-15-911) contains supplementary material, which is available to authorized users.

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The genome of the African trypanosome Trypanosoma brucei.

Matthew Berriman, Elodie Ghedin, Christiane Hertz-Fowler … (2005)

African trypanosomes cause human sleeping sickness and livestock trypanosomiasis in sub-Saharan Africa. We present the sequence and analysis of the 11 megabase-sized chromosomes of Trypanosoma brucei. The 26-megabase genome contains 9068 predicted genes, including approximately 900 pseudogenes and approximately 1700 T. brucei-specific genes. Large subtelomeric arrays contain an archive of 806 variant surface glycoprotein (VSG) genes used by the parasite to evade the mammalian immune system. Most VSG genes are pseudogenes, which may be used to generate expressed mosaic genes by ectopic recombination. Comparisons of the cytoskeleton and endocytic trafficking systems with those of humans and other eukaryotic organisms reveal major differences. A comparison of metabolic pathways encoded by the genomes of T. brucei, T. cruzi, and Leishmania major reveals the least overall metabolic capability in T. brucei and the greatest in L. major. Horizontal transfer of genes of bacterial origin has contributed to some of the metabolic differences in these parasites, and a number of novel potential drug targets have been identified.

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Continuous cultivation of Trypanosoma brucei blood stream forms in a medium containing a low concentration of serum protein without feeder cell layers.

H Hirumi, K Hirumi (1989)

Blood stream forms (BSF) of Trypanosoma brucei brucei GUT at 3.1 were propagated in vitro in the absence of feeder layer cells at 37 C, using a modified Iscove's medium (HMI-18). The medium was supplemented with 0.05 mM bathocuproine sulfonate, 1.5 mM L-cysteine, 1 mM hypoxanthine, 0.2 mM 2-mercaptoethanol, 1 mM sodium pyruvate. 0.16 mM thymidine, and 20% (v/v) Serum Plus (SP) (Hazleton Biologics, Lenexa, Kansas). The latter contained a low level of serum proteins (13 micrograms/ml). Each primary culture was initiated by placing 3.5-4 x 10(6) BSFs isolated from infected mice in a flask containing 5 ml of the medium (HMI-9) supplemented with 10% fetal bovine serum (FBS) and 10% SP. The cultures were maintained by replacing the medium every 24 hr for 5-7 days. During this period, many BSFs died. However, from day 4 onward, long slender BSFs increased in number. On days 5-7, trypanosome suspensions were pooled and cell debris was removed by means of diethylaminoethyl cellulose (DE52) column chromatography. Blood stream forms then were collected by centrifugation, resuspended in fresh medium at 7-9 x 10(5)/ml, and transferred to new flasks. Subcultures were maintained by readjusting the BSF density to 7-9 x 10(5)/ml every 24 hr. Concentrations of FBS were reduced gradually at 5-7-day intervals by alternating the amounts of FBS and SP in HMI-9 with 5% FBS and 15% SP, with 2% FBS and 18% SP, and finally with 20% SP (HMI-18). By this method, 2-3 x 10(6) VSFs/ml were obtained consistently every 24 hr. for more than 80 days.(ABSTRACT TRUNCATED AT 250 WORDS)

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Ribosome profiling reveals pervasive translation outside of annotated protein-coding genes.

Nicholas Ingolia, Gloria Brar, Noam Stern-Ginossar … (2014)

Ribosome profiling suggests that ribosomes occupy many regions of the transcriptome thought to be noncoding, including 5' UTRs and long noncoding RNAs (lncRNAs). Apparent ribosome footprints outside of protein-coding regions raise the possibility of artifacts unrelated to translation, particularly when they occupy multiple, overlapping open reading frames (ORFs). Here, we show hallmarks of translation in these footprints: copurification with the large ribosomal subunit, response to drugs targeting elongation, trinucleotide periodicity, and initiation at early AUGs. We develop a metric for distinguishing between 80S footprints and nonribosomal sources using footprint size distributions, which validates the vast majority of footprints outside of coding regions. We present evidence for polypeptide production beyond annotated genes, including the induction of immune responses following human cytomegalovirus (HCMV) infection. Translation is pervasive on cytosolic transcripts outside of conserved reading frames, and direct detection of this expanded universe of translated products enables efforts at understanding how cells manage and exploit its consequences. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

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Author and article information

Contributors

Bryan C Jensen: bryan.jensen@seattlebiomed.org

Gowthaman Ramasamy: gowthaman.ramasamy@seattlebiomed.org

Elton J R Vasconcelos: elton.vasconcelos@seattlebiomed.org

Nicholas T Ingolia: ingolia@berkeley.edu

Peter J Myler: mylerpj@u.washington.edu

Marilyn Parsons: mparsons@u.washington.edu

Journal

Journal ID (nlm-ta): BMC Genomics

Journal ID (iso-abbrev): BMC Genomics

Title: BMC Genomics

Publisher: BioMed Central (London )

ISSN (Electronic): 1471-2164

Publication date (Electronic): 20 October 2014

Publication date PMC-release: 20 October 2014

Publication date Collection: 2014

Volume: 15

Issue: 1

Electronic Location Identifier: 911

Affiliations

[ ]Seattle Biomedical Research Institute, 307 Westlake Ave N, Seattle, WA 98109-5219 USA

[ ]Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3202 USA

[ ]Department of Global Health, University of Washington, Harris Hydraulics Building, 1705 NE Pacific St #310E, Box 357965, Seattle, WA 98195 USA

[ ]Department of Biomedical Informatics and Medical Education, University of Washington, Seattle, WA 98195 USA

Article

Publisher ID: 6600

DOI: 10.1186/1471-2164-15-911

PMC ID: 4210626

PubMed ID: 25331479

SO-VID: 63bdca54-e56c-4aa9-b0d1-5a56651ae33c

License:

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

History

Date received : 21 September 2014

Date accepted : 9 October 2014

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ScienceOpen disciplines: Genetics

Keywords: ribosomal proteins,ribosome profiling,stage-regulation,trypanosome,translation

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ScienceOpen disciplines: Genetics

Keywords: ribosomal proteins, ribosome profiling, stage-regulation, trypanosome, translation

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

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Most cited references 56

The genome of the African trypanosome Trypanosoma brucei.

Continuous cultivation of Trypanosoma brucei blood stream forms in a medium containing a low concentration of serum protein without feeder cell layers.

Ribosome profiling reveals pervasive translation outside of annotated protein-coding genes.

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