On the extent and role of the small proteome in the parasitic eukaryote Trypanosoma brucei
1 Department of Epidemiology of Microbial Diseases, School of Public Health, Yale University, New Haven, CT, USA
2 Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried, Germany
3 Department of Cell Biology and Internal Medicine, School of Medicine, Yale University, New Haven, CT, USA
4 Current address: San Francisco General Hospital, Pulmonary & Critical Care, San Francisco, CA, USA
5 Current address: Institute of Molecular Biology gGmbH, Mainz, Germany
BMC Biology 2014, 12:14 doi:10.1186/1741-7007-12-14Published: 19 February 2014
Additional file 1:
Coding potential of the 987 transcripts described in Figure 1. Setting a lower limit of 25 aa, all the potential ORFs are listed. For example Tb1.NT.1_1 refers to the novel transcript Tb1.NT.1 according to the nomenclature by Kolev et al.  and _1 indicates ORF #1. The numbers in parenthesis specify the CDS.
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Additional file 2: Figure S1:
Length distribution of the predicted 4,699 ORFs. Figure S2. Growth analysis of the five small proteins with faster or slower growth in procyclics following induction of RNAi. Figure S3. Growth analysis of the seven small proteins essential in procyclics following induction of RNAi with and without expression of an RNAi-resistant construct. The data are based on three independent experiments. Figure S4. Northern blot analysis of essential ORFs. Northern blots for six (Tb11.NT.108, Tb10.NT.90, Tb10.NT.86, Tb10.NT.87, Tb11.NT.28 and Tb3.NT.18) of the seven essential transcripts. Marker sizes in kb are indicated on the left. The predicted transcript size is indicated below each panel. Northern blot analysis for Tb11.NT.29 can be found in Kolev NG, Franklin JB, Carmi S, Shi H, Michaeli S, Tschudi C: The transcriptome of the human pathogen Trypanosoma brucei at single-nucleotide resolution. PLoS Pathog 2010, 6:e1001090 . Figure S5. Semi-quantitative RT-PCR analysis of RNAi knockdown. RT-PCR was performed on the transcripts encoding each essential small protein from procyclic cells un-induced (-) or induced (+) for RNAi. Histone 4 (H4) was used as a control. The small protein transcript and H4 are indicated to the right of each panel. Figure S6. Listing of the seven essential proteins in procyclics with a C-terminal GFP or HA tag. All constructs rescued the lethal RNAi phenotype. Figure S7. Western blot analysis of GFP-tagged proteins encoded by an RNAi-resistant construct. Size markers (kDa) are shown at the left. Figure S8. Growth analysis of bloodstream-form cells following RNAi against four small proteins. Growth for un-induced (tet-) and induced (tet+) cells are shown in log scale. The data are based on three independent experiments. Figure S9. Listing of oligonucleotides used in this study.
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Additional file 3:
Listing of the 178 transcripts (sheet 178 ORFs) and 42 transcripts (sheet 42 ORFs) encoding potential ORFs with matching peptides and hits in Kinetoplastids. Panigrahi et al.: A comprehensive analysis of Trypanosoma brucei mitochondrial proteome. Proteomics 2009, 9:434–450 . Butter 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. Mol Cell Proteomics 2013, 12:172–179 . viv, T. vivax; cru, T. cruzi; Leish, Leishmania; con, T. congolense; evansi, T. evansi. PTM Abbreviations: P, phosphorylation; S, sumoylation; PM, palmitoylation; G, glycosylation. Phosphorylation prediction: NetPhos http://www.cbs.dtu.dk/services/NetPhos/ webcite; Sumoylation prediction: http://sumosp.biocuckoo.org/ webcite; Palmitoylation Prediction: http://csspalm.biocuckoo.org/ webcite; Glycosylation Prediction: http://www.cbs.dtu.dk/services/YinOYang/ webcite.
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Additional file 4:
T. brucei predicted ORFs conserved in representative eukaryotes. BLAST bit scores of T. brucei predicted ORFs with potential homologs in representative eukaryotes. Bit scores are shown after the accession number and they represent a normalized version of the raw BLAST alignment score.
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Additional file 5:
Survey of conserved domains in 173 T. brucei predicted ORFs.
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