Comparative ribosome profiling reveals extensive translational complexity in different Trypanosoma brucei life cycle stages

While gene expression is a fundamental and tightly controlled cellular process that is regulated at multiple steps, the exact contribution of each step remains unknown in any organism. The absence of transcription initiation regulation for RNA polymerase II in the protozoan parasite Trypanosoma brucei greatly simplifies the task of elucidating the contribution of translation to global gene expression. Therefore, we have sequenced ribosome-protected mRNA fragments in T. brucei, permitting the genome-wide analysis of RNA translation and translational efficiency. We find that the latter varies greatly between life cycle stages of the parasite and ∼100-fold between genes, thus contributing to gene expression to a similar extent as RNA stability. The ability to map ribosome positions at sub-codon resolution revealed extensive translation from upstream open reading frames located within 5′ UTRs and enabled the identification of hundreds of previously un-annotated putative coding sequences (CDSs). Evaluation of existing proteomics and genome-wide RNAi data confirmed the translation of previously un-annotated CDSs and suggested an important role for >200 of those CDSs in parasite survival, especially in the form that is infective to mammals. Overall our data show that translational control plays a prevalent and important role in different parasite life cycle stages of T. brucei.

[1]  Nicholas T Ingolia,et al.  Genome-wide translational profiling by ribosome footprinting. , 2010, Methods in enzymology.

[2]  J. Donelson,et al.  Different trans RNA splicing events in bloodstream and procyclic Trypanosoma brucei. , 2008, Molecular and biochemical parasitology.

[3]  R. Jackson,et al.  The mechanism of eukaryotic translation initiation and principles of its regulation , 2010, Nature Reviews Molecular Cell Biology.

[4]  M. Carrington,et al.  Developmentally regulated instability of the GPI-PLC mRNA is dependent on a short-lived protein factor , 2005, Nucleic acids research.

[5]  P. Stadler,et al.  RNA Maps Reveal New RNA Classes and a Possible Function for Pervasive Transcription , 2007, Science.

[6]  M. Selbach,et al.  Global quantification of mammalian gene expression control , 2011, Nature.

[7]  Jonathan E. Allen,et al.  Genome sequence of the human malaria parasite Plasmodium falciparum , 2002, Nature.

[8]  R. Wek,et al.  Reinitiation involving upstream ORFs regulates ATF4 mRNA translation in mammalian cells. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[9]  Nicholas T. Ingolia,et al.  Ribosome Profiling of Mouse Embryonic Stem Cells Reveals the Complexity and Dynamics of Mammalian Proteomes , 2011, Cell.

[10]  M. Mann,et al.  Comparative Proteomics of Two Life Cycle Stages of Stable Isotope-labeled Trypanosoma brucei Reveals Novel Components of the Parasite's Host Adaptation Machinery* , 2012, Molecular & Cellular Proteomics.

[11]  C. Clayton,et al.  The role of the 5'-3' exoribonuclease XRNA in transcriptome-wide mRNA degradation. , 2011, RNA.

[12]  A. Hehl,et al.  A conserved stem-loop structure in the 3' untranslated region of procyclin mRNAs regulates expression in Trypanosoma brucei. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[13]  Nicholas T. Ingolia,et al.  Genome-Wide Analysis in Vivo of Translation with Nucleotide Resolution Using Ribosome Profiling , 2009, Science.

[14]  Zefeng Wang,et al.  Glycolysis modulates trypanosome glycoprotein expression as revealed by an RNAi library , 2002, The EMBO journal.

[15]  J. Bonfield,et al.  Finishing the euchromatic sequence of the human genome , 2004, Nature.

[16]  C. Keasar,et al.  Cap-binding activity of an eIF4E homolog from Leishmania. , 2004, RNA.

[17]  E. Marcotte,et al.  Absolute protein expression profiling estimates the relative contributions of transcriptional and translational regulation , 2007, Nature Biotechnology.

[18]  M. Moore.  From Birth to Death: The Complex Lives of Eukaryotic mRNAs , 2005, Science.

[19]  G. Cross,et al.  Systematic Study of Sequence Motifs for RNA trans Splicing in Trypanosoma brucei , 2005, Molecular and Cellular Biology.

[20]  Juan Pablo Couso,et al.  Peptides Encoded by Short ORFs Control Development and Define a New Eukaryotic Gene Family , 2007, PLoS biology.

[21]  Graziano Pesole,et al.  uAUG and uORFs in human and rodent 5'untranslated mRNAs. , 2005, Gene.

[22]  K. Matthews.  Controlling and Coordinating Development in Vector-Transmitted Parasites , 2011, Science.

[23]  K. Matthews,et al.  Post-transcriptional control of nuclear-encoded cytochrome oxidase subunits in Trypanosoma brucei: evidence for genome-wide conservation of life-cycle stage-specific regulatory elements , 2006, Nucleic acids research.

[24]  A. Hinnebusch,et al.  Phosphorylation of initiation factor 2α by protein kinase GCN2 mediates gene-specific translational control of GCN4 in yeast , 1992, Cell.

[25]  J. Hoheisel,et al.  Heat shock causes a decrease in polysomes and the appearance of stress granules in trypanosomes independently of eIF2α phosphorylation at Thr169 , 2008, Journal of Cell Science.

[26]  David Fenyo,et al.  Four histone variants mark the boundaries of polycistronic transcription units in Trypanosoma brucei. , 2009, Genes & development.

[27]  R. Brun,et al.  Cultivation and in vitro cloning or procyclic culture forms of Trypanosoma brucei in a semi-defined medium. Short communication. , 1979, Acta tropica.

[28]  E. Ullu,et al.  Temporal order of RNA-processing reactions in trypanosomes , 1993 .

[29]  G. Cross,et al.  Utilization of amino acids by Trypanosoma brucei in culture: L-threonine as a precursor for acetate , 1975, Parasitology.

[30]  A. Hinnebusch.  Gene‐specific translational control of the yeast GCN4 gene by phosphorylation of eukaryotic initiation factor 2 , 1993, Molecular microbiology.

[31]  M. Ronen,et al.  Multiple levels of gene regulation mediate differentiation of the intracellular pathogen Leishmania , 2011, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[32]  J. Weissman,et al.  Selective Ribosome Profiling Reveals the Cotranslational Chaperone Action of Trigger Factor In Vivo , 2011, Cell.

[33]  Bernadette A. Thomas,et al.  Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study 2010 , 2012, The Lancet.

[34]  International Human Genome Sequencing Consortium.  Finishing the euchromatic sequence of the human genome , 2004 .

[35]  Alan D. Lopez,et al.  The Global Burden of Disease Study , 2003 .

[36]  C. Clayton,et al.  Post-transcriptional regulation of gene expression in trypanosomes and leishmanias. , 2007, Molecular and biochemical parasitology.

[37]  B. Barrell,et al.  A Re-Annotation of the Saccharomyces Cerevisiae Genome , 2001, Comparative and functional genomics.

[38]  Miguel A. Andrade-Navarro,et al.  uORFdb—a comprehensive literature database on eukaryotic uORF biology , 2013, Nucleic Acids Res..

[39]  R. Kiss,et al.  Galectin-1: a small protein with major functions. , 2006, Glycobiology.

[40]  John D. Storey,et al.  Genome-wide analysis of mRNA translation profiles in Saccharomyces cerevisiae , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[41]  Keith R. Matthews,et al.  Differential Trypanosome Surface Coat Regulation by a CCCH Protein That Co-Associates with procyclin mRNA cis-Elements , 2009, PLoS pathogens.

[42]  David Horn,et al.  Single-locus targeting constructs for reliable regulated RNAi and transgene expression in Trypanosoma brucei. , 2008, Molecular and biochemical parasitology.

[43]  Xuning Wang,et al.  Genome-wide analysis of mRNA abundance in two life-cycle stages of Trypanosoma brucei and identification of splicing and polyadenylation sites , 2010, Nucleic acids research.

[44]  Alejandro Sanchez-Flores,et al.  High-throughput phenotyping using parallel sequencing of RNA interference targets in the African trypanosome. , 2011, Genome research.

[45]  A. Djikeng,et al.  A new twist in trypanosome RNA metabolism: cis-splicing of pre-mRNA. , 2000, RNA.

[46]  S Kobayashi,et al.  Small Peptides Switch the Transcriptional Activity of Shavenbaby During Drosophila Embryogenesis , 2010, Science.

[47]  Han-kuei Huang,et al.  GTP hydrolysis controls stringent selection of the AUG start codon during translation initiation in Saccharomyces cerevisiae. , 1997, Genes & development.

[48]  D. Horn.  Codon usage suggests that translational selection has a major impact on protein expression in trypanosomatids , 2008, BMC Genomics.

[49]  E. Fèvre,et al.  The Burden of Human African Trypanosomiasis , 2008, PLoS neglected tropical diseases.

[50]  Steven L Salzberg,et al.  Fast gapped-read alignment with Bowtie 2 , 2012, Nature Methods.

[51]  Paul Lasko,et al.  Translational control in cellular and developmental processes , 2012, Nature Reviews Genetics.

[52]  Gerald A Tuskan,et al.  Discovery and annotation of small proteins using genomics, proteomics, and computational approaches. , 2011, Genome research.

[53]  A. Aggarwal,et al.  mRNA Regulation by Puf Domain Proteins , 2006, Science's STKE.

[54]  A. von Haeseler,et al.  A Developmentally Regulated Aconitase Related to Iron-regulatory Protein-1 Is Localized in the Cytoplasm and in the Mitochondrion of Trypanosoma brucei * , 2000, The Journal of Biological Chemistry.

[55]  M. Mann,et al.  Comprehensive mass-spectrometry-based proteome quantification of haploid versus diploid yeast , 2008, Nature.

[56]  M. Ouellette,et al.  A combined proteomic and transcriptomic approach to the study of stage differentiation in Leishmania infantum , 2006, Proteomics.

[57]  M. Shapira,et al.  Evolutionary Conservation and Diversification of the Translation Initiation Apparatus in Trypanosomatids , 2012, Comparative and functional genomics.

[58]  Kenneth Stuart,et al.  Transcription of Leishmania major Friedlin chromosome 1 initiates in both directions within a single region. , 2003, Molecular cell.

[59]  Jef D Boeke,et al.  Functional genomics of genes with small open reading frames (sORFs) in S. cerevisiae. , 2006, Genome research.

[60]  J. Donelson,et al.  Differential expression of a protease gene family in African trypanosomes. , 2009, Molecular and biochemical parasitology.

[61]  M. Shapira,et al.  Binding Specificities and Potential Roles of Isoforms of Eukaryotic Initiation Factor 4E in Leishmania , 2006, Eukaryotic Cell.

[62]  P. Myler,et al.  Transcription Initiation and Termination on Leishmania major Chromosome 3 , 2004, Eukaryotic Cell.

[63]  C. Clayton,et al.  Mechanisms of developmental regulation in Trypanosoma brucei: a polypyrimidine tract in the 3'-untranslated region of a surface protein mRNA affects RNA abundance and translation. , 1997, Nucleic acids research.

[64]  S. Kramer,et al.  Developmental regulation of gene expression in the absence of transcriptional control: the case of kinetoplastids. , 2012, Molecular and biochemical parasitology.

[65]  C. Clayton,et al.  DRBD1 is the Trypanosoma brucei homologue of the spliceosome-associated protein 49. , 2009, Molecular and biochemical parasitology.

[66]  S. Beverley,et al.  Coupling of poly(A) site selection and trans-splicing in Leishmania. , 1993, Genes & development.

[67]  Tim R. Mercer,et al.  Differentiating Protein-Coding and Noncoding RNA: Challenges and Ambiguities , 2008, PLoS Comput. Biol..

[68]  Shulamit Michaeli,et al.  The Transcriptome of the Human Pathogen Trypanosoma brucei at Single-Nucleotide Resolution , 2010, PLoS pathogens.

[69]  Barbara M. Bakker,et al.  Glycolysis in Bloodstream Form Trypanosoma brucei Can Be Understood in Terms of the Kinetics of the Glycolytic Enzymes* , 1997, The Journal of Biological Chemistry.

[70]  Nicholas T. Ingolia,et al.  Ribosome Profiling Provides Evidence that Large Noncoding RNAs Do Not Encode Proteins , 2013, Cell.

[71]  B. A. Castilho,et al.  Novel Membrane-Bound eIF2α Kinase in the Flagellar Pocket of Trypanosoma brucei , 2007, Eukaryotic Cell.

[72]  Anne E Willis,et al.  A perspective on mammalian upstream open reading frame function , 2013, The International Journal of Biochemistry & Cell Biology.

[73]  Isabel M. Vincent,et al.  The threonine degradation pathway of the Trypanosoma brucei procyclic form: the main carbon source for lipid biosynthesis is under metabolic control , 2013, Molecular microbiology.

[74]  G. Hong,et al.  Nucleic Acids Research , 2015, Nucleic Acids Research.

[75]  N. Standart,et al.  Translation initiation in Leishmania major: characterisation of multiple eIF4F subunit homologues. , 2005, Molecular and biochemical parasitology.

[76]  J. Galagan,et al.  Evolutionary roles of upstream open reading frames in mediating gene regulation in fungi. , 2009, Annual review of microbiology.

[77]  Tamir Tuller,et al.  Determinants of Translation Elongation Speed and Ribosomal Profiling Biases in Mouse Embryonic Stem Cells , 2012, PLoS Comput. Biol..

[78]  David M. A. Martin,et al.  The Genome of the African Trypanosome Trypanosoma brucei , 2005, Science.

[79]  V. Mootha,et al.  Upstream open reading frames cause widespread reduction of protein expression and are polymorphic among humans , 2009, Proceedings of the National Academy of Sciences.

[80]  C. Clayton,et al.  Trypanosoma brucei PUF9 Regulates mRNAs for Proteins Involved in Replicative Processes over the Cell Cycle , 2009, PLoS pathogens.

[81]  L. Cui,et al.  The Puf-family RNA-binding protein PfPuf2 regulates sexual development and sex differentiation in the malaria parasite Plasmodium falciparum , 2010, Journal of Cell Science.

[82]  M. Kozak,et al.  Selection of initiation sites by eucaryotic ribosomes: effect of inserting AUG triplets upstream from the coding sequence for preproinsulin. , 1984, Nucleic acids research.

[83]  A. Frasch,et al.  RNA-Binding Domain Proteins in Kinetoplastids: a Comparative Analysis , 2005, Eukaryotic Cell.

[84]  M. Mann,et al.  Andromeda: a peptide search engine integrated into the MaxQuant environment. , 2011, Journal of proteome research.

[85]  W. Huber,et al.  which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. MAnorm: a robust model for quantitative comparison of ChIP-Seq data sets , 2011 .

[86]  Mark D. Robinson,et al.  edgeR: a Bioconductor package for differential expression analysis of digital gene expression data , 2009, Bioinform..

[87]  E. Ullu,et al.  A common pyrimidine-rich motif governs trans-splicing and polyadenylation of tubulin polycistronic pre-mRNA in trypanosomes. , 1994, Genes & development.

[88]  Eileen Kraemer,et al.  EuPathDB: The Eukaryotic Pathogen database , 2012, Nucleic Acids Res..

[89]  R. Simon,et al.  Signaling of cell fate decisions by CLAVATA3 in Arabidopsis shoot meristems. , 1999, Science.

[90]  B. A. Castilho,et al.  Novel membrane-bound eIF2alpha kinase in the flagellar pocket of Trypanosoma brucei. , 2007, Eukaryotic cell.

[91]  Anna M. McGeachy,et al.  The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA fragments , 2012, Nature Protocols.

[92]  M. Parsons,et al.  Changes in polysome profiles accompany trypanosome development. , 1998, Molecular and biochemical parasitology.

[93]  A. Furger,et al.  Elements in the 3' untranslated region of procyclin mRNA regulate expression in insect forms of Trypanosoma brucei by modulating RNA stability and translation , 1997, Molecular and cellular biology.

[94]  G. Cross,et al.  Histone H3 trimethylated at lysine 4 is enriched at probable transcription start sites in Trypanosoma brucei. , 2010, Molecular and biochemical parasitology.

[95]  Terry K. Smith,et al.  Regulation of Trypanosoma brucei Total and Polysomal mRNA during Development within Its Mammalian Host , 2013, PloS one.

[96]  M. Tomita,et al.  Bioinformatic analysis of post‐transcriptional regulation by uORF in human and mouse , 2007, FEBS letters.

[97]  Tanaka.  The role of , 2000, Journal of insect physiology.

[98]  M. Mann,et al.  MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification , 2008, Nature Biotechnology.

[99]  J. Donelson,et al.  The Genome of the African Trypanosome , 2002 .

[100]  Martin Kircher,et al.  Deep proteome and transcriptome mapping of a human cancer cell line , 2011, Molecular systems biology.

[101]  G. Cross,et al.  Gene expression in Trypanosoma brucei: lessons from high-throughput RNA sequencing. , 2011, Trends in parasitology.

[102]  L. Farinelli,et al.  Spliced Leader Trapping Reveals Widespread Alternative Splicing Patterns in the Highly Dynamic Transcriptome of Trypanosoma brucei , 2010, PLoS pathogens.

[103]  Antonin Morillon,et al.  Pervasive transcription constitutes a new level of eukaryotic genome regulation , 2009, EMBO reports.

[104]  J. Rinn,et al.  Peptidomic discovery of short open reading frame-encoded peptides in human cells , 2012, Nature chemical biology.