Nonsense-Mediated Decay in Trypanosomes Figure 1. Effects of alterations in UPF1 expression on growth, interaction with UPF2, and localisation. (A) Procyclic trypanosomes with or without tetracycline-inducible TbUPF1-FLAG or Tbupf1-1-FLAG transgenes (induced for 48 h), or inducible RNAi hp-hairpin (induced for 72 h), p2T7 – opposing T7 promoters (induced for 24 h) were grown with (+) or without (2) tetracycline (200 ng/ml) for the indicated time and then harvested for RNA preparation. Northern blots were probed for TbUPF1 or tubulin (TUB) RNA. The extra band in lane 6 is the dsRNA synthesised from TbUPF1 RNAi plasmid and the extra bands in lanes 8 and 10 are the exogenous expressed TbUPF1/Tbupf1-1 transcripts. (B) Procyclic cells containing hp RNAi or over-expression plasmids were grown in presence and absence of tetracycline (0.1 ug/ml) and the cell count taken every 24 hours for at least 7 days. Cells were diluted to 56105 cells/ml to maintain exponential growth. Cumulative growth curves were plotted using Kaleidograph, and the doubling times for each line in the presence and absence of tetracycline were estimated using the computer-fitted growth curves. (C) Growth curve of procyclic trypanosome containing the p2T7 UPF1 RNAi construct. Details of the growth conditions and induction are as in (B). (D) Western blot analysis on 26107 cells of bloodstream or procyclic trypanosomes expressing flag-tagged wild-type TbUPF1 or the helicase mutant Tbupf1-1, using anti-flag antibody. The control antibody was anti-aldolase for bloodstream forms; for procyclics a cross-reacting band is shown. (E) Interaction of tagged TbUPF1 with TbUPF2. TbUPF2-myc (lanes 7–12) was expressed in trypanosomes with a V5-tagged TbUPF1 gene. Cells with no myc-tagged protein (lanes 1–6) served as a control. Extracts were immunoprecipitated with anti-myc, in the presence or absence of RNase. 2.5% of the input (lanes 1, 2, 7, 8) and unbound (lanes 3, 4, 9, 10,) fractions, and the whole eluates (lanes 5, 6, 11, 12,) were separated by SDS-PAGE, blotted, and probed with antibodies to myc, V5 and PABP. *IgG heavy chain. (F) TbUPF1-flag is in the cytoplasm. Fixed procyclic trypanosomes expressing UPF1-FLAG were stained for the flag tag and counterstained for DNA with DAPI. Cells with no FLAG construct showed only much fainter background fluorescence. doi:10.1371/journal.pone.0025112.g001 inducibly-expressed TbUPF1-flag was readily detectable by immunofluorescence and located predominantly in the cytoplasm (Figure 1F). TbUPF1 interacts with TbUPF2 To find out whether TbUPF1 and TbUPF2 interact, we co- expressed TbUPF2-myc in cells with in situ V5-tagged TbUPF1 (Figure 1E). Immunoprecipitation with anti-myc antibody resulted in co-precipitation of both V5-TbUPF1 and PABP (lane 11). The interaction between V5-TbUPF1 and TbUPF2-myc was not affected by RNase treatment, whereas PABP was released (lane 12). Comparison of the band intensities in lanes 7 and 8 (2.5% of input) and pull down (100%) indicated that only a small proportion of V5-TbUPF1 was bound to TbUPF2-myc, but the myc pull-down also showed poor efficiency (compare lanes 9 & 10 with lanes 7 & 8). The control immunoprecipitation (using cells that did not express a myc-tagged protein) pulled down very little V5-TbUPF1 and no detectable PABP (lanes 5 & 6). These results confirmed that Tb11.02.4270 is indeed likely to be TbUPF2, as predicted in [55]. The results also indicated that tagged TbUPF2 and TbUPF1 were bound to PABP in an RNA-dependent fashion. So far, our results were consistent with a role of TbUPF1 in cytoplasmic mRNA metabolism. PLoS ONE | www.plosone.org 3 September 2011 | Volume 6 | Issue 9 | e25112