between the termination codon and poly(A) binding protein). In trypanosomes, option (1) seems unlikely to be important for the CAT reporters: the relevant region was of prokaryotic origin. Any mechanism depending on recognition of the splice junction via the EJC is unlikely. Some of the EJC proteins are present, and form a complex, in trypanosomes [66], though neither binding to the exon junction nor a role in trans splicing have been confirmed. Even assuming that the EJC is deposited at the trans splice junction, it would be stripped off by the scanning small ribosomal subunit complex before translation initiation. An involvement of the trans splice site per se cannot, however, be ruled out. Splicing- dependent, EJC-independent NMD was shown in S. pombe [27]: notably, NMD was stimulated by the presence of an intron upstream of a PTC – as would occur in trypanosomes. S. pombe splicing-dependent NMD was however stronger than the NMD- like effects that we saw in trypanosomes. In a recent study of mRNP composition, UPF1 was found in all mRNPs examined, but the amount was greater in mRNAs that are susceptible to NMD, and also correlated positively with the length of the 39-UTR [18]. In addition, even low amounts of translational read-through or reinitiation reduced NMD. Our experiments with reporters yielded very mixed evidence concerning a role of the 39- UTR length in determining trypanosome mRNA abundance. For the WT-CAT ORF, we saw a clear decrease in CAT mRNA abundance after insertion of an ATG-less GFP sequence in the 39- UTR. The result was, however, contradicted using the LPTC-CAT ORF, where the same insertion had no effect (Figure 4D). The most recent evidence suggests that at a global level, 39-UTR length negatively affects mRNA half-lives in mouse cells [67], but the effect is both variable and weak. In trypanosomes, deep sequencing data reveals no such correlation (T. Manful, A Fadda and C. Clayton, ZMBH, manuscript submitted). This is not surprising. Trypanosome (and mouse) 39-UTRs are somewhat longer than those of yeast: for trypansoomes, the median length is 400 nt, and 39-UTRs over 1 kb are by no means unusual [68]. The heavy reliance of trypanosomes on regulation of mRNA stability to control gene expression may have driven the evolution of long 39-UTRs: these will have space for multiple elements which can be recognized by regulatory sequence- or secondary-structure- specific RNA-binding proteins. Tethering of PABP to reporter mRNAs had a very strong stabilizing effect. This is may well be unrelated to NMD - in yeast, even a non-translated mRNA can be stabilized by tethering of PABP [62]; 59 decapping is inhibited. It was intriguing that we saw effects of PABP1, but not PABP2. In the related parasite Leishmania, both proteins are cytoplasmic and can associate with eIF4G in vitro, but using cytoplasmic extracts, only PABP1 was shown to copurify with eIF4G [69]. It is therefore quite possible that in trypanosomes, also, tethered PABP1 inhibits 59-39 degradation by binding to the cap-bound translation initiation complex. Whether this also stimulates translation, as in yeast [70] remains to be determined. Our results leave open the question of the function of UPF1 in trypanosomes. It is likely to play some role in Kinetoplastids since the gene is well conserved throughout Kinetoplastids. TbUPF1 must also be bound to some RNAs, since it was found to interact not only with UPF2, but also, in an RNA-dependent fashion, with PABP. In addition, tethering of UPF1 to an mRNA did increase the degradation rate, and there were various RNAi effects on reporters that appeared to be independent of the presence of a PTC. Depletion of TbUPF1 did not affect the T. brucei steady-state mRNA transcriptome as judged by microarray analysis; prelim- inary results from an RNA-Seq study confirm this (T. Manful, ZMBH, unpublished). There was also little evidence for a Nonsense-Mediated Decay in Trypanosomes requirement of either UPF1 or UPF2 in trypanosome growth and division. It is therefore possible that UPF1 affects mRNA in conditions or life-cycle stages that were not examined in our study, or has other functions. Overall it is not clear that classical NMD exists in trypanosomes. It would not be required for disposal of pseudogene mRNAs: most trypanosome pseudogenes are either located in telomeric regions that are not transcribed, or produce transposon-related RNAs that are disposed of by the RNAi machinery [71,72]. Alternative trans splicing can definitely create short upstream ORFs and bicistronic mRNAs, but their low abundance could easily be caused simply by low utilization of the alternative processing sites. One more set of observations is worthy of comment: inhibition of translation, either by adding a secondary structure, or increasing the length of the 59-UTR, decreased the amount of reporter mRNAs. Although recent results from yeast have shown that loss of ribosomes is not an obligatory prerequisite for mRNA decay [73], translation initiation certainly must stop before 59-39 degradation can begin. Both our results, and those from S. pombe [27] would be consistent with a link between the average number of ribosomes on an mRNA and its stability. Assuming that initiation is to some degree stochastic, the lower the average number of ribosomes on an ORF is, the higher the proportion of mRNAs with no ribosomes at all. Our observations therefore fit with the idea that release of an mRNA from polysomes can render it more susceptible to degradation. Methods Trypanosomes, cell culture, and plasmid construction Procyclic and bloodstream trypanosomes of the Lister 427 strain, and bloodstream-form parasites of the AnTat 1.1 strain, were cultured according to standard protocols. Procyclic trypano- somes were diluted to a density of 56105/ml before the density reached 86106/ml; bloodstream cells were diluted to 16105/ml before the density reached 16106/ml. Unless noted, experiments were done using cells expressing the Tet repressor from pHD1313 [74]. In some cases the cells also expressed T7 polymerase. Tetracycline-dependent transcription by RNA polymerase 1 or the T7 polymerase was induced by addition of 0.1–1 mg/ml tetracycline. Selecting antibiotics were always removed at least one day before experiments were initiated. All plasmid constructs are described in Table S1 and the oligonucleotides in Table S2. For RNA interference, dsRNA was either expressed as a stem-loop, transfected into the standard line, or using opposing T7 promoters with a line expressing T7 polymerase in addition to the tet repressor [74]. Plasmids were linearized, transfected into the parasites by electroporation, and permanent cloned cell lines were obtained by limiting dilution [75]. Western blotting Cells (56106 to 26107) were harvested by centrifugation and the pellets boiled in reducing SDS sample buffer before separation by SDS-PAGE. After Western blotting, tags were detected with the following primary antibodies at 1:2000 dilution: mouse M2 (anti-Flag, Sigma), mouse anti-V5 (Invitrogen), mouse anti-myc (Santa Cruz Laboratories) and, for the TAP tag, peroxidase anti- peroxidase (PAP, Sigma). Detection was done using the ECL kit (GE Healthcare). In order to visualise TbUPF1 in trypanosomes, we raised antibody against N-terminally truncated TbUPF1 (residues 396–842), but the serum failed to detect the protein on Western blots. PLoS ONE | www.plosone.org 10 September 2011 | Volume 6 | Issue 9 | e25112