Etheridge_Fig. S1 NTD * * (368) (154) (296) (131) (146) (477) (271) (391) (141) (248) CTDCTD (570) (363) (483) (232) (340) (675) (446) (588) (318) (422) Figure S1 Partial multiple sequence alignment of trypanosomal TUTases and kinetoplast poly(A) polymerase 1. Multiple sequence alignments were performed with the T-coffee algorithm (Notredame et al. 2000) and refined manually. Protein sequences used in the alignment are: TbRET1 (AAK38334), TbRET2 (AAO63567), TbTUT3 (XP_822966), TbTUT4 (DQ923393) and TbKPAP1 (EF650028). Identical amino acids are white on black, and similar ones are black on grey background. The discontinuous N-terminal (NTD) domain is boxed, the middle domain (MD) is denoted by a dotted line, and the “CTD” brackets indicate the carboxyterminal domain. The signature motif of the Pol  nucleotidyl transferase superfamily is underlined. Catalytic aspartate residues of the metal-binding triad are shown by asterisks. Amino acid position mutated to alanine in KPAP1 is indicated by a triangle. Residues in TbTUT4 involved in uracil base recognition are marked by arrows and those implicated in RNA binding are shown by filled diamonds.

Initial Depolarized 0h24h 48h 72h 96h 120h Unstained 2913 Stained 2913 CCCP Control fluorescence SSC Etheridge_Fig. S2 Figure S2 Mitochondrial membrane potential in KPAP1-depleted cells. At indicated RNAi induction time points, cells were treated with membrane potential-sensitive fluorescent dye, MitoTracker Red CMX ROS, or CCCP protonophore. Red fluorescence was measured by flow cytometry.

Etheridge_Fig. S3 (1) (81) (82)(162) (163) (243) (244) (325) PCR with A357-A358 A482 A483 A484 A485 (1) (82) (163) (81) (162) (221) PCR with A359-A360 A478 A479 A605 Oligo Northern Probe Nested cRT-PCR 1 st Round Nested cRT-PCR 2 nd Round RPS12 Pre-Edited mRNA RPS12 Edited mRNA AATAAGATATGTTTT Figure S3 Positioning of Northern blotting probes and cRT-PCR primers for pre-edited and edited RPS12 mRNAs. Extended DNA probes for Northern hybridization were PCR-amplified with indicated primers.

Etheridge_Fig. S4 parental Pre-edited Edited 1 st Editing Block Pre-edited KPAP1 RNAi 3' Truncation Edited Random editing Figure S4 Partial multiple sequence alignments of RPS12 mRNAs amplified with primers specific for the pre-edited transcript. Alignment was performed with ClustalW and refined manually. Conservative positions are black on green background.