Immunofluorescence staining Approximately 106 cells were collected by centrifugation and resuspended in 50 ml of PBS. Cells were fixed in 4% paraformal- dehyde in PBS for 20 min, washed twice in PBS and allowed to settle onto poly-lysine-coated slides. Cells were permeabilised in 0.2% Triton-X100 in PBS, washed twice and blocked in 0.5% gelatine in PBS for 1 hour. Primary antibody was added at the manufacturer-recommended dilution, incubated for 1 hour and the slides washed 3 times in PBS before addition of Alexa -594- conjugated secondary antibody (1 hour). Slides were washed, stained with DAPI (10 min), and washed twice more before drying the slide and mounting with Vectorshield. Northern blotting and RNA half-life assay Parasites were harvested at room temperature and RNA isolated by Trizol extraction. RNA was denatured with formamide and formaldehyde, separated on a denaturing formaldehyde gel, blotted onto Nytran and hybridized. The DNA probes were made from plasmids or PCR products, by random priming with 32P label dCTP. For RNA half-life assays, synthesis and maturation of mRNA were inhibited by addition to the growth medium of 2.0 mg/mL Sinefungin then, 5 min later, 10 mg/mL Actinomycin D [59,76]. The time of Actinomycin D addition to the cell was taken as time 0. Cells were collected at various times and RNA isolated by Trizol extraction. RNA levels were estimated by Northern blotting using [32P]-labelled probes, and quantitated by phosphorimaging. The 7SLRNA, or mRNAs encoding histone H4 (HISH4) or beta- tubulin (TUB) were used as loading controls. To obtain half-lives, we used the phosphorimaging results starting 2 h after Actinomy- cin D addition. Half-lives were measured for each individual experiment and, if there were at least three experiments, expressed as arithmetic mean 6 standard deviation. For two experiments an average is given. Transcriptome analysis Microarray analyses to compare the RNA of wild-type and TbUPF1-depleted procyclic forms (cell line with RNAi targeting the entire TbUPF1 ORF) were done using 6 slides, with two biological replicates, as described in [77]. Almost no differences were found, so the results have not been uploaded into a public database. Real-time PCR Real time PCR was performed using the SYBR green I kit (Roche Applied Science). RNA was isolated using Trizol. About 1 mg total RNA was DNase treated, then cDNA was synthesised using SuperScript III (Invitrogen). After RNaseH digestion of the DNA-RNA hybrid, the cDNA was diluted and used for real time PCR in the LightCyclerR 480 system. Standard curves for each primer set - CZ 3634 and CZ 3633 for CAT and CZ 3636 and CZ 3635 for the reference neomycin phosphotransferase (NEO) gene were included during each run to calculate the in-run PCR efficiency. Primers were designed using Primer3 software [78]. Relative quantification was done according to the E (efficiency) method using the LightCyclerR 480 software (Roche Applied Science). Co-immunoprecipitation Procyclic cell lines carrying V5-tagged TbUPF1, either alone or with co-expressed TbUPF2-myc, were induced with 100 ng/ml tetracycline for 48 hours in medium lacking selective antibiotics. For each sample, 66108 cells were harvested and suspended in Nonsense-Mediated Decay in Trypanosomes 2 ml of lysis buffer containing 20 mM Tris-HCl pH 7.4, 10 mM NaCl, 0.1% IGEPAL, 100 nM okadaic acid and Complete protease Inhibitor without EDTA (Roche). The cells were lysed by passing through a 21-gauge syringe several times on ice. Cell lysis was monitored under the microscope. The salt concentration was adjusted to 150 mM NaCl, and the lysate clarified by centrifugation at 13,000 rpm, 20 min at 4uC (microfuge). The cleared lysates (1 ml each) were treated with to RNase A (200 mg/ mL) or 4 mM Vanadyl Ribonucleoside complexes (VRCs, Sigma). About 800 mL of each lysate (6 RNase A) was incubated with 50 mL of Myc-agarose (Sigma) for 2 h at 4uC with rotation. Immune complexes were washed 3 times with 500 mL of 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.1% IGEPAL, and 100 nM okadaic acid, boiled in 80 mL of 26 SDS sample buffer, then analyzed by Western blotting. For the input and flow-through fractions, 2.5% of the total (equivalent to 66106 cells) was loaded. The Western blot was probed with anti-V5, anti-myc or anti- PABP antibodies (gift from Dr. Laurie Read). Supporting Information Figure S1 Sequence alignment of TbUPF1 with UPF1s from other species. Residues that are100% conserved are in solid red boxes, while those in which at least 70% are similar are outlined in red. The arginine residue at position 747 in the trypanosome sequence was mutated to make upf1-1. Other arginines in this region that were shown to be essential in yeast or human UPF1 are indicated by asterisks. The Figure was generated by Clustalw2 and ESPript. Sequences used were: Tb: Trypanosoma brucei CP000068.1. Sc: Saccharomyces cerevisiae NC_001145.2. At: Arabidopsis thaliana AF484122.1. Tv: Trichomonas vaginalis DS113229.1. Dd: Dictyostelium discoideum XM_631398.1. Gi: Giardia intestinalis DQ861427.1. Tg: Toxoplasma gondii XM_002368483.1. Pf: Plasmodium falciparum AE014185.2. Hs: Homo sapiens NM_002911.3. (PDF) Figure S2 The strategy for replacement of the GPI-PLC gene with a mutant copy. In wild-type cells the GPI-PLC gene (pink) is flanked by HSP100 (green) and ß9-COP genes. The poly(A) site is marked as ‘‘a’’ and the two alternative spliced learder addition sites as black dots (‘‘SL’’ for ‘‘spliced leader’’). The panel below shows the loci in the double knockout line, and below that the construct that was used to return the full-length or PTC versions of GPI-PLC. The bottom panel shows the loci in cells containing the integrated return construct. (PDF) Figure S3 RNA degradation kinetics for selected CAT constructs, including the effects of UPF1 RNAi, UPF2 RNAi, or tethering of PABP. Results are shown as arithmetic mean 6 standard deviation for 3 or more experiments, with all values at +2 h set as 100%. When two experiments were done, each is shown individually. The curves were fitted for time points from +2 h onwards. The half-lives shown were calculated separately for each individual experiment and – if there were three experiments - are presented as arithmetic mean 6 standard deviation. The constructs used are indicated above the graphs; the presence of a tethered protein or induction of RNAi are also indicated. (PDF) Figure S4 Tethering of UPF1 to T7-transcribed CAT-B- ACT mRNA increases the mRNA degradation rate. The Figure show the results for two inependent experiments, done using the clones shown in Figure 5C, and as described for Figure PLoS ONE | www.plosone.org 11 September 2011 | Volume 6 | Issue 9 | e25112