Nonsense-Mediated Decay in Trypanosomes Figure 3. Effect of ORF length on mRNA levels from a CAT reporter. (A) Map of the CAT-NEO reporter plasmid used to analyse requirements for NMD. spRRNA: non-transcribed portion of rRNA spacer, used for integration into the genome; PRRNA – rRNA promoter; 59: 59-UTR; 39: 39-UTR; TER – symbol for termination codon. This plasmid was used in procyclic forms. For bloodstream forms, spRRNA and PRRNA were replaced with a segment from the tubulin locus. (B) Effect of the position of the termination codon on the CAT/NPT ratio in procyclic trypanosomes, with pol I transcription of the reporter. The cartoons on the left illustrate the CAT mRNAs investigated. SL: spliced leader; GFP: complete or partial GFP coding region. The 59-UTR of the WT mRNA is 70 nt, the CAT ORF 660 nt and the 39-UTR 326 nt. The graphs on the right show the ratio of CAT to NPT mRNA, normalised to wild-type, expressed as a percentage. Results are arithmetic mean and standard deviation for at least three biological replicates. Asterisks indicate cloned cell lines, the rest of the results are for populations. (C) Effect of ORF length and UPF1 RNAi on CAT/NPT mRNA ratio in bloodstream trypanosomes, with pol II transcription of the reporter. Results are for 3–4 independent cloned lines, each measured once or twice, and are expressed as arithmetic mean 6 standard deviation. The arithmetic mean CAT/NPT ratio for WT-CAT was set to 100%. Columns 2,3 and 5,6 are for cell lines containing a hairpin UPF1 RNAi plasmid, without (2, 5) or with (3, 6) tetracycline. (D) Results are the same as in (C), but each cell line was considered individually, with the CAT/NPT value minus tetracycline set to 100%. (E) Effect of TbUPF1 RNAi on levels of CAT mRNAs in procyclic forms. Trypanosomes with in situ V5-tagged UPF1 were transfected with reporter plasmids and the hpTbUPF1 RNAi plasmid. Tetracycline was added where indicated, for 48 h. The ratio of CAT to NPT mRNA was measured by real-time PCR, and expressed as a percentage of wild-type. Results are arithmetic mean and standard deviation for at least three biological replicates. The Western blots beneath the graph show expression of V5-UPF1, with aldolase (ALD) as loading control. The V5 signals were normalised using the aldolase band, and expressed as a percentage relative to lane 1. The high V5 signal in the LPTC RNAi line might be due to tagging of both alleles. The double band of V5-TbUPF1 was rarely seen; it might be caused by phosphorylation [79]. doi:10.1371/journal.pone.0025112.g003 locus in bloodstream-form trypanosomes. Results from four independent cell lines yielded EPTC-CAT mRNA levels of between 8% and 59% relative to full-length CAT (Figure 3D, compare bars 1 and 4). This shows that the effect of the PTC was independent of both the transcribing polymerase and the trypanosome life-cycle stage. Effect of RNAi targeting TbUPF1 on reporter mRNAs We now investigated whether TbUPF1 was required for the relatively low abundance of the EPTC-CAT mRNA. We made cell lines that expressed WT-CAT, LPTC-CAT or EPTC-CAT mRNA and had inducible TbUPF1 RNAi. The levels of CAT mRNA were measured with or without induction of RNAi. For bloodstream PLoS ONE | www.plosone.org 5 September 2011 | Volume 6 | Issue 9 | e25112