forms, expressing WT-CAT, using RNA polymerase II, we assayed 3 independent cloned lines. The CAT mRNA levels in the RNAi lines showed considerable variation between clones (Figure 3C, lanes 2,3,5,6). To measure the effect of RNAi, we therefore compared individual clones with and without tetracycline. For three EPTC-CAT lines, the increase in CAT mRNA was 1.3–1.7 fold; however similar increases were seen in 3 out of 4 WT-CAT lines (RNA levels changed 0.5–1.9 fold) (Figure 3D). Thus depletion of UPF1 did not specifically reverse the effect of the PTC on reporter mRNA abundance. Preliminary results for UPF2 RNAi revealed no effect at all on either mRNA (not shown). In the procyclic cell line with the WT-CAT gene (polymerase I transcription) and the TbUPF1 RNAi plasmid, TbUPF1 RNAi caused a 30–40% decrease in WT-CAT mRNA abundance (Figure 3E, lanes 3 & 4). With the LPTC-CAT transgene, results were similar (lanes 7 & 8). Addition of tetracycline to cells with no RNAi construct had no effect (Figure 3E, lanes 2, 6 and 10). In the cell line with the EPTC-CAT transgene, the increase in CAT mRNA was unaffected by RNAi induction (Figure 3D lanes 11 &12). From all experiments, so far we therefore concluded that introduction of PTCs – or decreasing the ORF length - reduced the abundances of the CAT and GPI-PLC mRNAs in trypano- somes, but there was no convincing evidence for a role of UPF1 in destruction of EPTC-CAT mRNA. The length of the 39-UTR does not reproducibly affect reporter mRNA abundance Our results in Figure 2 and 3B were consistent with the prediction that mRNA degradation increases with the distance of the termination codon from the poly(A) tail. To find out whether 39-UTR length really was important, we extended the 39UTR of the WT-CAT mRNA and measured the relative mRNA abundance, using procyclic trypanosomes. Inserting 190 nt of GFP sequence (39+WT), or even a complete GFP ORF (720 nt, 39++WTs) had no effect on WT-CAT mRNA levels (Figure 4A). Correct processing of the mRNA was confirmed by Northern blotting (Figure 4B). Since the 39-UTR GFP insertion was immediately downstream of the WT-CAT termination codon, potentially allowing reinitia- tion, we created a new plasmid in which the 720 nt GFP ORF had been mutated so that it lacked initiation codons (GFPns, GFP-no- start). The abundance of mRNA (39++WT) from this plasmid was 55% of WT, and the effect was reversed to 82% by RNAi against TbUPF1 (Figure 4C). This was consistent with 39-UTR-dependent and UPF1-dependent NMD. We were, however, unable to confirm this result with the LPTC-CAT and EPTC-CAT ORFs. Lengthening their 39UTRs by 720 nt, using the ATG-less GFP (from 393 to 1112 nt in LPTC-CAT and 743 to 1463 nt in EPTC- CAT) did not decrease CAT mRNA levels (Figure 4A); for EPTC- CAT, results stayed between 35% and 40% even with the longest extension. When we put together the results of all these reporter experiments, the length of the 39-UTR, over a 330 nt–1.2 kb range, had no consistent effect on trypanosome mRNA abundance (Figure 4D). If there is a 39-UTR length effect at all, it must be overridden even by minor differences in the 39-UTR sequence. Tethering of poly(A)-binding protein inhibits trypanosome mRNA decay In mammals, budding yeast and Drosophila, tethering of cytoplasmic poly(A)-binding protein (PABP) downstream of a PTC inhibits NMD [61,62]. It was thought that the tethered PABP specifically antagonised NMD by effectively shortening the 39-UTR [61], but recently the effect has been shown to be more Nonsense-Mediated Decay in Trypanosomes general and not dependent on PTC recognition [62]. To find out whether the PABP effect was seen in trypanosomes, we inserted six ‘‘B’’ boxes downstream of EPTC-CAT, or downstream of GFPns in the 39++WT construct (Figure 4E). This lengthened each 39-UTR by 270 nt but had no effect on mRNA abundance (compare Figures 4A and 4E). Proteins bearing a lambda N peptide at the N- terminus, and a myc tag at the C-terminus, were inducibly expressed in cell lines expressing the new mRNAs and the effect on CAT mRNA was measured. All three proteins were expressed at comparable levels. There are two PABPs in trypanosomes, PABP1 (Tb09.211.0930) and PABP2 (Tb09.211.2150). Tethering of either GFP or PABP2 to the EPTC-CAT-box B mRNA had no effect on mRNA abundance. In contrast, tethering of PABP1 to either 39++WT or EPTC-CAT mRNA – decreasing the stop codon - PABP distances to 720 and 100 nt respectively - indeed increased mRNA levels. PABP1 tethering caused complete stabilization of the mRNAs (Figure 4E and Figure S3G, H). Changing the 59-UTR length decreases mRNA abundance and inhibits translation We next wanted to check whether the physical length of the ORF or the distance from the 59-end to the stop codon, influenced mRNA abundance. We therefore extended the 59-UTRs of WT- CAT, LPTC-CAT and EPTC-CAT by inserting ATG-less GFP sequences of 190 nt (59+), 720 nt (59++) or 1440 nt (59+++). Results are shown in Figure 5A and Northern blots to verify RNA sizes are in Figure 4B. Lengthening the 59-UTR in front of the WT-CAT or LPTC-CAT ORFs decreased the mRNA levels by 20– 50%. In contrast, the amount of CAT mRNA from the EPTC- CAT constructs increased steadily as the length of the 59-UTR increased; with the longest 59-UTR, rescue was complete, since the 59++EPTC mRNA level was the same as that of the 59++WT mRNA. Thus the level of mRNA was not consistently increased in parallel with the distance of the termination codon from the 59- end. NMD depends on the recognition of the nonsense mutations by the translation machinery. It was therefore important to examine the effects of 59-UTR extension on translation. To do this, using WT-CAT cell lines, we measured amounts of CAT protein by Western blotting, then normalised them to the amount of CAT mRNA from Figure 5A. Insertion of GFPns in the 59-UTR strongly inhibited translation whereas the same sequence in the 39-UTR did not (Figure 5B). Thus we could not see whether the effects we had seen were due to 59-UTR length per se, or to translation inhibition. To distinguish the effects of translation inhibition from those related to 59-UTR length, we sought a method to inhibit translation alone. Treatment of trypanosomes with protein synthesis inhibitors increases the levels of many mRNAs – including the WT-CAT mRNA used in this study (not shown). The reasons have not been defined, but a requirement for unstable protein factors for mRNA decay (e.g. [57]), or effects on mRNA processing [63] have been suggested (reviewed in [48]). To analyse translation alone, we therefore inserted a translation-inhibitory hairpin into the 59-UTRs of the reporter plasmids (control data for translation shown later). This decreased the abundances of the WT-CAT and LPTC-CAT mRNAs (Figure 5C) in the same way as lengthening the 59-UTR (Figure 5A). This indicates that translation favours mRNA maintenance. In contrast, the amount of hp-EPTC-CAT mRNA was similar to that of EPTC-CAT mRNA (Figure 5C). The simplest interpretation of this result is that EPTC- CAT mRNA has low abundance because of NMD, while the hp- EPTC-CAT and hp-CAT mRNAs have low abundance because they have no ribosomes on the ORF. PLoS ONE | www.plosone.org 6 September 2011 | Volume 6 | Issue 9 | e25112