the splice junction [2,7,23]. The EJC contains the NMD factor Upf3; this recruits Upf2 which in turn recruits Upf1. In contrast, in Schizosaccharomyces pombe, unspliced mRNAs were subject to weak NMD: the abundance of a reporter mRNA decreased concor- dantly with coding region length, and 39-UTR length had little influence [27]. The presence of a nearby intron, either upstream or downstream of the PTC, enhanced NMD and the EJC was not required [27]. NMD targets mRNAs that have short open reading frames (ORFs) upstream of the start codon [28,29,30], suppresses splicing defects [28,31,32,33] and disposes of non-coding polyadenylated RNAs [34]. To investigate the overall role of NMD in regulation of gene expression, transcriptome profiles have been obtained for cells depleted of NMD factors. In S. cerevisiae, nearly a tenth of all mRNAs showed increased abundance in Dupf1 cells, [35,36,37]. The identified transcript set was markedly skewed towards mRNAs that had relatively low abundances, and about half of the affected mRNAs were bound to Upf1, suggesting that they were direct NMD targets [38]. Depletion of Upf1 in animals caused 2–10 fold increases in many mRNAs, a substantial proportion of which are likely to be direct NMD substrates [31,37,39]. In Caenorhabditis elegans, for example, depletion of Upf1 increased the abundance of mRNAs that arose from splicing errors, or had upstream open reading frames [28]. Despite all this, the full physiological importance of NMD is not yet clear. Notably, Upf1, Upf2 and Upf3 are not essential for viability in yeast or Caenorhabditis elegans. Upf1 is essential for shoot development in Arabidopsis [40], mammalian embryonic viability [41] and Drosophila development [42]; but it is not known whether NMD-specific or non-NMD-specific function(s) of Upf1 are implicated. So far, the only protist in which NMD has been investigated is Giardia lamblia. In Giardia, introductions of PTCs into a luciferase reporter mRNA reduced mRNA abundance by up to 70% [13]. The dependence of the PTC-induced mRNA decrease on Upf1 was not tested, but extra expression of tagged Upf1 reduced the abundance of the truncated luciferase mRNA further and influenced a small number of native mRNAs [43]. Kinetoplastids are protists that diverged from animals and plants early in eukaryotic evolution. In Kinetoplastids, transcrip- tion is polycistronic. Individual mRNAs are cleaved from the precursor by trans splicing of a 39 nt capped leader to the 59 end of the RNA, and by polyadenylation at the 39-end; only one cis- spliced mRNA has been experimentally demonstrated [44]. The abundances of mRNAs are determined post-transcriptionally and variations in mRNA half-lives have been extensively documented. In most cases examined, mRNA abundances were shown to be determined by sequences in the 39-UTR and regulatory RNA- binding proteins [45,46,47]. The mRNA decay machinery is overall similar to that in yeast, plants, and animals [48,49,50,51], and RNA interference is present [52], but the existence of NMD has hitherto not been documented. In this paper we investigate the function of Upf1 and the possible existence of NMD in the Kinetoplastid Trypanosoma brucei. Results Sequence analysis and domain organisation of TbUPF1 and TbUPF2 The trypanosome genome has single loci encoding TbUPF1 (Tb927.5.2140) [14] and TbUPF2 (Tb11.02.4270). The TbUPF1 homolog encodes a protein of predicted molecular weight of 93.3 kDa which shares 42.77% and 43.58% identity with human and S. cerevisiae Upf1, respectively (see Figure S1). It has a highly conserved N-terminal Cysteine-Histidine-rich domain (CH do- Nonsense-Mediated Decay in Trypanosomes main) which includes the amino acid residues needed for interacting with Upf2 [14] – present in all other sequences analysed except that from Giardia intestinalis. The amino acid sequence from residue 368 to 608 of TbUPF1 contains the ATPase and DEAD-like helicase domains that are known to be essential for NMD. TbUPF2 is a protein of 162.5 kDa. The N-terminal part contains two MIF4G-like domains with very low scores (InterPro) followed by a single consensus MIF4G domain (SMART score 0.0013), analogous to the third MIF4G domain of human Upf2. There is then a Glu/Asp-rich acidic region. We identified no matches for Upf3, Smg-6 or Smg-7. BLASTp using Smg-1 revealed a variety of kinase domains but no clear homologue. Effect of TbUPF1 and TbUPF2 depletion on trypanosome growth To find out whether TbUPF1 and TbUPF2 were required for trypanosome growth, we created bloodstream and procyclic trypanosome cell lines with tetracycline-inducible RNAi. We failed to generate an appropriate anti-UPF1 antibody, so to assess the effect of the RNAi on the protein, we tagged one copy of UPF1 in situ, so that the expressed protein had an N-terminal V5 tag. Two procyclic form clones with up to 88% reduction in the mRNA (Figure 1A) showed slightly prolonged division times but this was independent of the presence of tetracycline (Figure 1B), so might be due to clonal variation. A procyclic line in which dsRNA against TbUPF1 was made by synthesising the entire ORF as a double strand, using opposing T7 polymerase promoters, grew slowly in the absence of tetracycline and almost completely stopped dividing upon tetracycline addition (Figure 1C). This could have been a consequence of extremely effective depletion, but off-target effects cannot be ruled out. In bloodstream trypanosomes with one V5-tagged UPF1 allele, RNAi targeting UPF1 resulted in over 95% depletion of V5- UPF1 without any effect on growth (not shown). Inducible RNAi against TbUPF2 did not affect procyclic trypanosome growth or morphology up to 7 days post-tetracycline addition (Figure 1B) although the transcript was 80% reduced after 3 days (not shown). In a recently published high-throughput RNAi screen, cells with UPF1 or UPF2 RNAi even appeared to have a selective advantage in bloodstream forms, but not procyclic forms [53]: although high-throughput results must always be viewed with caution, in this case they were consistent with the results of our focussed experiments. All evidence so far therefore indicates that neither UPF1 nor UPF2 is required for bloodstream trypanosome growth and division, but there were hints that some UPF1 might be needed for optimal growth of procyclic forms. We do not know whether either protein is completely dispensable because a knockout was not attempted. For over-expression analysis, we created bloodstream and procyclic trypanosomes with tetracycline-inducible expression of FLAG-tagged wild type TbUPF1. We also inducibly expressed an R747C mutant version, Tbupf1-1. This is equivalent to the yeast R779C and human R844C Upf1 mutants, expression of which abrogated NMD in a dominant-negative fashion [12,54]. Expression of either protein in bloodstream forms had no effect on growth. In procyclic forms, the TbUPF1-flag and Tbupf1-1-flag transcripts were, respectively, ,2-fold and 13-fold more abun- dant than the endogenous TbUPF1 transcript (Figure 1A). Correspondingly, Tbupf1-1-flag protein was considerably more abundant than TbUPF1-flag (Figure 1D). Expression of TbUPF1- flag marginally inhibited cell growth; expression of Tbupf1-1-flag resulted in some slightly abnormal parasite morphology (not shown) but did not change the division time (Figure 1B). The PLoS ONE | www.plosone.org 2 September 2011 | Volume 6 | Issue 9 | e25112