The abundance of a PTC-containing trypanosome mRNA decreases with ORF length To find out whether trypanosomes degrade mRNAs containing premature termination codons (PTCs) we inserted PTCs into a trypanosome ORF. We started with bloodstream trypanosomes lacking the non-essential gene (Tb927.2.6000) encoding glycosyl phosphatidylinositol phospholipase C (GPI-PLC) [56]. We took plasmids containing either the wild-type GPI-PLC gene or a PTC- containing version, with an associated selectable marker. These plasmids were transfected into trypanosomes so that they would integrated into the original locus [57] (Figure S2). The wild-type open reading frame contains 342 codons; the modified transgenes contained PTCs at codon 17, codon 102 or codon 237. We had previously reported that the PTC at position 17 (51 nt ORF) had little effect on the amount of mRNA, but the abundance had not been quantified [57]. More careful analysis revealed that this PTC reduced the steady state level of the GPI-PLC mRNA to 35% of normal (Figure 2). PTCs at codons 102 or 237 reduced the levels marginally, to ,80% of normal. These cells lines contain most of the drug resistance markers available in trypanosomes. To determine the role of TbUPF1 in the GPI-PLC mRNA decay, we attempted to deplete TbUPF1 mRNA by transient transfection of morpholino oligonucleotides; this had little effect but the depletion was insufficiently effective to draw any conclusions. The abundance of a PTC-containing reporter mRNA decreases concordantly with ORF length To test for NMD in procyclic-form T. brucei, we used a reporter containing the chloramphenicol acetyl transferase (CAT) ORF followed by the 39-UTR of the actin gene (CAT-ACT construct). Downstream of this was the neomycin phosphotransferase (NEO) gene, with 59 and 39-UTRs (Figure 3A). We transfected linearized plasmid into procyclic trypanosomes, and selected for G418 resistance. The plasmid was designed to integrate into the silent rDNA spacer, with an rRNA promoter driving transcription by RNA polymerase I. Using this system, the level of CAT mRNA could be measured using NEO RNA as an internal control for both transcription rate and plasmid copy number. Use of pol I gave us sufficient RNA for quantification and detection on Northern blots, Figure 2. Shortening the GPI-PLC ORF length reduces mRNA abundance. (A) Northern blots of RNA extracted from two indepen- dent clones containing GPI-PLC transgenes, wild type (342 codons, lanes 2,3) or with PTCs at codons 17 (lanes 4,5) or 102 (lanes 6,7) or 237 (lanes 8,9). RNA extracted from a GPI-PLC +/2 cell line is shown for comparison in lane 1. (B) Quantitation of GPI-PLC mRNA by phosphorimager, plotted onto a map of the open reading frame. The values are averages of measurements from the two clones and are shown relative to the wild type transgene, after normalization for loading using rRNA. The GPI-PLC +/2 cells gave a relative mRNA abundance of 0.91. doi:10.1371/journal.pone.0025112.g002 Nonsense-Mediated Decay in Trypanosomes enabling us to confirm that mRNAs had the correct size (shown later). We note that in trypanosomes, any effect of trans splicing on mRNA decay should be seen equally in mRNAs made by RNA polymerase I or RNA polymerase II, since the evidence so far indicates that transcripts made by either polymerase are processed with equal efficiency [58]. All measurements were made in cells that had been growing for at least 2 days without G418. The CAT ORF is of prokaryotic origin. To find out if a nonsense-mediated decay effect could be seen, we inserted PTCs at various positions. The CAT termination codon is at nt 660 (WT- CAT, WT660 in Figure 3B). Insertion of 4 nt at position 218 resulted in a frame shift, giving a TGA termination codon at position 243 and a second TAG 5 codons downstream. This construct was named ‘‘early PTC’’ (EPTC-CAT) and yielded no detectable CAT protein (judged by Western blotting, not shown). Translation reinititation is unlilkely with this construct since the next ATG after the PTC is 45 nt downstream. A similar insertion at position 519 gave a TGA at position 591 (late PTC, LPTC-CAT) (Figure 3B); in this case two more in-frame termination codons are 9 and 10 codons downstream, with two ATGs within 18 nt of the PTC; should any translation re-initiation occur, the longest possible ORF would extend only 54 nt beyond the WT-CAT termination codon. In addition to the EPTC-CAT and LPTC-CAT ORFs, we inserted segments of the GFP gene at various positions to give ORF lengths of 393, 963 and 1380 nt. Cell lines were generated and we confirmed that the mRNA sizes were as expected (not shown). We then measured the level of CAT mRNA by reverse transcription and real-time PCR (Figure 3B). ORFs of 243–393 nt (EPTC243, EPTC249, EPTC393 in the Figure) all yielded 30–40% of wild- type RNA, while the 1380 nt CAT-GFP ORF doubled the amount of mRNA relative to WT-CAT. Taken together these results were consistent with three hypotheses: either the introduction of a PTC per se decreased mRNA abundance, or the length of the ORF or the 39-UTR was the determining factor. To follow this up, we made a construct with a 963 nt CAT-GFP ORF, but the same 39- UTR as the EPTC-CAT mRNA. The resulting EPTC-CAT-GFP mRNA level was (65% of WT) intermediate between that of EPTC and WT-CAT. This suggests that long ORFs favour mRNA abundance, while the presence of the untranslated CAT segment has a negative influence. The results do not show whether the effect of the untranslated CAT segment is sequence-specific, or is related to the overall length of the 39-UTR. To measure mRNA half-lives, we treated the cells with Sinefungin and Actinomycin D to halt transcription, and then measured mRNA levels by Northern blot. We consistently observed, as seen previously for experiments using Actinomycin D alone [59], that the apparent mRNA levels either stayed constant, or increased over the first 2 h after inhibitor addition (Figure S3A), so in general we calculated half-lives starting from the 2 h time-point. Pooling results for all cell lines with normal levels of UPF1 and UPF2 (including RNAi lines with no tetracycline induction, a total of at least 8 measurements for each CAT construct), we found that the half-lives of the WT-CAT and EPTC-CAT mRNAs were 2.460.2 h and 1.860.5 h respectively. The result for WT-CAT agrees with the previous estimate made using Actinomycin D alone [60]. The half-life numbers were, however, so strongly dependent on which time points were used to plot the decay curves that it was impossible to make any quantitative judgements from the results. To make sure that the results with the reporter were not artefacts of pol I transcription, and to find out whether PTC introduction affected mRNA abundance in bloodstream forms, we integrated the WT-CAT and EPTC-CAT ORFs into the tubulin PLoS ONE | www.plosone.org 4 September 2011 | Volume 6 | Issue 9 | e25112