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1 SUPPLEMENTARY INFORMATION doi: /nature12864 Supplementary Table Peak Gene code Screen Function or Read analysis AMP reads camp annotation reads minor Tb AMP ISWI Confirmed A Tb camp MEK Confirmed, 2 hits B Tb AMP ubiquitin activating Confirmed 1 hit enzyme, C Tb camp AMPK SNF1 like family Confirmed 1 hit, could also target Tb D Tb camp hypothetical Confirmed 1 hit Tb both phosphatase 1, Confirmed, several hits E Tb both phosphatase 1, confirmed, several hits Tb both phosphatase 1, confirmed, several hits Tb both phosphatase 1, adjacent gene F Tb camp phosphoglycerate mutase G T camp Variant Surface Glyco, H Confirmed: has a discrete peak from PP1 confirmed Lacks one primer site: artefact Tb both adenosine, confirmed, several hits Tb both adenosine confirmed, several hits I Tb camp GMP synthase confirmed J Tb camp dual specificity Confirmed, 2 hits phosphatase minor Tb camp Hypothetical confirmed K Tb camp confirmed, several hits (HYP 2) L Tb both ADSL Confirmed, several hits M Tb camp Confirmed Tb camp Artefact (primer binding) Sequence due to artefactual primer binding N Tb camp Artefact (primer binding) Sequence due to artefactual primer binding O Tb both Pentatricopeptide Lacks one primer site: artefact P Tb AMP adenylate, Confirmed, though one primer site very weak Tb camp NEK Confirmed, several hits Q Tb camp NEK Confirmed, several hits Tb camp NEK Confirmed, several hits Tb camp RNA-binding Confirmed, 2 hits R Tb camp WW domain Not confirmed- no unique hit S Tb both YAK Confirmed, 2 hits T Tb camp IMP dehydrogenase Confirmed, several hits Tb camp phosphatase and tensin Confirmed, cannot be distinguished from Tb U Tb camp pre-mrna-splicing factor CWC26 like C terminus minor Tb camp DNA repair, Confirmed, cannot be distinguished from Tb Confirmed, second hit targets Tb intergenic region Tb camp Protein phosphatase 2C Confirmed, second hit targets Tb intergenic region
2 RESEARCH SUPPLEMENTARY INFORMATION V Tb camp GRESAG 4 receptor adenylate cyclase intergenic region Lacks one primer site: artefact minor Tb camp Conserved hypothetical Confirmed minor Tb camp Conserved hypothetical Confirmed W Tb both ADSS Confirmed, several hits X Tb camp A regulatory subunit Confirmed Tb both Confirmed, several (hyp1) hits Y Z Tb both Tb AMP Mitochondrial SSU ribosomal, Not confirmed, bystander for Tb Confirmed; cannot distinguish these two genes. Intergenic region only Tb AMP Trichohyalin, Confirmed; cannot distinguish these two genes. Intergenic region only Supplementary Table 1 List of genes identified after selection with 100µM pcptcamp or 10µM 8- pcpt-2 -O-Me-5 -AMP. The labeled major peaks are annotated on Figure 1c and the genes at the corresponding genomic location annotated in column 2. Column 3 indicates whether the genes were selected in either or both selective screens (pcptcamp indicated by camp ; 8-pCPT-2 -O-Me-5 -AMP indicated by AMP ), whereas column 4 provides a functional prediction for each, where available. Column 5 indicates a summary of the analysis of the relevant Ion Torrent TM reads for that gene, with Columns 6 and 7 providing the total number of reads that aligned to the open reading frame from each selection. 2
3 SUPPLEMENTARY INFORMATION RESEARCH Supplementary Table Gene Screen Function Tested Slow Growth? SIF Res? RITSeq 6 DIF PCF Tb AMP ISWI ND ND Tb camp MEK ND Tb AMP ubiquitin activating 1 ND enzyme, Tb camp AMPK SNF1 like family 1 ND Tb camp hypothetical ND ND Tb both phosphatase 1, 2 Tb both phosphatase 1, 2 Tb both phosphatase 1, 2 Tb both phosphatase 1, ND ND adjacent gene Tb camp phosphoglycerate ND ND mutase Tb both adenosine, 3 Tb both adenosine 3 Tb camp GMP synthase 3 Tb camp dual specificity 4 phosphatase Tb camp Hypothetical ND Tb camp 4 (HYP 2) Tb both ADSL 3 3 Tb camp ND ND Tb camp NEK 4 ND ND Tb camp NEK 4 Tb camp NEK 4 Tb camp RNA-binding 5 Tb both YAK Tb camp IMP dehydrogenase ND ND Tb camp phosphatase and tensin 1 ND Tb camp pre-mrna-splicing ND ND factor CWC26 like C terminus Tb camp DNA repair, ND ND Tb camp Protein phosphatase 2C ND ND Tb camp Conserved hypothetical ND ND Tb camp Conserved hypothetical ND ND Tb both ADSS 3 3 Tb camp A 3 3 regulatory subunit Tb both (hyp1) Tb AMP Mitochondrial SSU ND ND ribosomal, Tb AMP Trichohyalin, ND ND 3
4 RESEARCH SUPPLEMENTARY INFORMATION Supplementary Table 2 Refined list of genes isolated from each selection regimen after removal of predicted artefactual hits. Column 1 shows the relevant gene ID, column 2 the screen from which it was selected (pcptcamp indicated by camp ; 8-pCPT- 2 -O-Me-5 -AMP indicated by AMP ), column 3 shows its predicted function or annotation. The genes tested in independent monomorphic or pleomorphic RNAi lines are shown in column 4 and their growth characteristics upon RNAi induction shown in column 5. Where pleomorphic lines were isolated, their capacity for stumpy formation is indicated in column 6, with SIF res? indicating whether each cell line showed SIF resistance and hence reduced stumpy formation in vivo. Columns 7 and 8 show whether a reduced fitness was observed for each gene in differentiating or procyclic form cells when analysed by genome wide phenotyping for loss or gain of fitness 20. Column 5 and 6 Notes: 1. No verified RNAi competent lines were isolated from several transfections. 2. Slow growth in vitro and, to a lesser extent, in vivo 3. Strong growth inhibition 4. Weak growth inhibition in vitro or in vivo 5. Leaky RNAi in all six lines tested. (Columns 7 and 8) Data derived from Alsford, S. et al. High-throughput phenotyping using parallel sequencing of RNA interference targets in the African trypanosome. Genome research, doi:gr [pii] 4
5 SUPPLEMENTARY INFORMATION RESEARCH Supplementary Table 3 A B C D E F Panther/Prosite/Interpro domains mrna expression profile Protein expression Phospho site BSF phosphorylation enriched (>10x) PCF phosphorylation enriched (>10x) Gene Predicted Function SL (1) INT (1) ST (1) SS/SL (2) PCF/BSF (3) Phosphorylation (4) Tb ISWI transcriptional silencer (5) ISWI CHROMATIN- REMODELING COMPLEX ATPASE ISW1 (PTHR10799:SF73) ND Tb MEK MITOGEN-ACTIVATED KINASE KINASE KINASE (PTHR24361) Tb Tb Tb Tb Tb Tb ubiquitin activating enzyme, AMPK SNF1 like family hypothetical phosphatase 1 phosphatase 1 phosphatase 1 NEDD8-ACTIVATING ENZYME E1 REGULATORY SUBUNIT (AMYLOID PROTEIN- BINDING PROTEIN 1) (AMYLOID BETA PRECURSOR PROTEIN- BINDING PROTEIN 1, 59 KDA)(APP-BP1) (PTHR10953:SF29) CARBON CATABOLITE- DEREPRESSING PROTEIN KINASE (PTHR24343:SF15) No domains identified; detected in the T. brucei flagellum proteome SERINE/THREONINE (PTHR11668:SF110) SERINE/THREONINE (PTHR11668:SF110) SERINE/THREONINE (PTHR11668:SF110) ND S486, S492, S713, S724, S747, S773, S854, T ND ND ND ND 1.49 S482, S486, T167, Y ND 0.45 ND ND ND 1.08 ND ND ND ND Tb None ND ND Tb dual specificity phosphatase DUAL SPECIFICITY (PTHR10159) ND ND S237, S307, S312, S513, S839 Tb ND 0.54 Tb Tb Tb Tb ) Tb Tb Tb Tb Tb Tb (HYP 2) NEK NEK NEK RNA-binding RBP7 RNA-binding RBP7 YAK Prokaryotic dksa C4-type zinc finger signature and profile (PROSITE PDOC00846); SSF52540 P- loop containing nucleoside triphosphate hydrolases ND S258 None ND ND ND SERINE/THREONINE- PROTEIN KINASE NEK (PTHR24362) SERINE/THREONINE- PROTEIN KINASE NEK (PTHR24362) SERINE/THREONINE- PROTEIN KINASE NEK (PTHR24362) Eukaryotic RNA Recognition Motif (RRM) profile (PS50102); FAMILY NOT Eukaryotic RNA Recognition Motif (RRM) profile (PS50102); FAMILY NOT DUAL SPECIFICITY PROTEIN KINASE YAK1 (PTHR24058:SF17) S2, S3, T195, T S2, T195, T ND S ND ND ND ND ND ND S112,S117,S23,S25,S28 S77, Y
6 RESEARCH SUPPLEMENTARY INFORMATION Tb Tb RNA-binding RBP7 YAK Eukaryotic RNA Recognition Motif (RRM) profile (PS50102); FAMILY NOT DUAL SPECIFICITY PROTEIN KINASE YAK1 (PTHR24058:SF17) ND ND ND ND S112,S117,S23,S25,S28 S77, Y868 Tb Tb ) Tb Tb ) Tb Tb Tb Tb ) Tb Tb ) Tb Tb ) Tb Tb ) (6) Tb Tb ) Tb Tb Tb Tb PTEN pre-mrnasplicing factor CWC26 like C terminus DNA repair helicase/ chromatin regulator PP2C Hypothetical Hypothetical A regulatory subunit (HYP1) Annotated Triohyalin, PROTEIN-TYROSINE PHOSPHATASE MITOCHONDRIAL 1 (PTHR10159:SF5) Bud13: Pre-mRNA-splicing factor of RES complex (PF09736) DNA REPAIR HELICASE RAD5,16 (PTHR10799:SF62) SWI/SNF-RELATED MATRIX-ASSOCIATED ACTIN-DEPENDENT REGULATOR OF CHROMATIN SUBFAMILY- RELATED (PTHR10799) 2C (PTHR13832) ND ND ND ND S75, S ND ND ND ND None ND ND S63 None ND PROTEIN KINASE A REGULA S2, S181, S218 SUBUNIT (PTHR11635:SF6) None ND ND ND Mitochondrial SSU ribosomal, ; KRIPP ND ND ND None ND ND Supplementary Table 3 Interpro analysis, mrna, and phospho-site data for each of the curated hits from the genome-wide RNAi screen for QS signal regulators. Column A shows the Gene ID for each curated molecule, Column B shows its predicted function, Column C shows the Interpro scan ( annotation for that gene, Column D shows the relative transcript expression profile for that gene in Slender, Intermediate and Stumpy forms derived from Digital-SAGE analysis, Column E shows the expression profile in slender and stumpy forms, or bloodstream and procyclic forms determined by SILAC analysis (Log2 fold change) and Column F shows the position and developmental regulation of phosphorylation sites for each encoded. Source data and further information 1. Capewell et al. PLoS One 2013 Jun 26;8(6):e Print Gunasekera et al. BMC Genomics Oct 16;13: Urbaniak et al., PLoS One. 2012;7(5):e Urbaniak et al., J. Proteome Res., 2013, 12 (5), pp Hughes et al.; EMBO J (9): S. Kramer, PhD thesis, University of Munich. 6
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