Nonsense-Mediated Decay in Trypanosomes Figure 6. Tethering of TbUPF1 decreases the abundance of CAT-ACT mRNA. (A) Reporter mRNAs with ACT 39-UTRs. used for tethering experiments, not to scale. For details see text and Table 1. The mRNAs were produced from a T7 promoter, in cells expressing T7 polymerase. (B) Fusion proteins used for tethering. For details see text and Table 1. (C) Northern blot showing CAT-ACT mRNAs in cells inducibly expressing GFP and TbUPF1 fusion proteins. Appropriate Western blots are beneath. Expression of tagged TbUPF1 or GFP was induced with 1 mg/ml tetracycline for 2 days. A portion of the cells was used for western analysis and the rest was used for total RNA isolation. Northern blots were probed with full length CAT probe and SRP was used as loading control. For western analysis, cell lines expressing the TbUPF1 with a flag tag were probed with anti-flag antibody and those with the TAP tag were probed with PAP antibody. The same blots were probed with anti-aldolase antibody as a loading control. (D) Quantitation of the data in (C). CAT mRNA amounts are expressed as the percentage of the appropriate control - lane 1 in (C). Results are the arithmetic mean of and standard deviation for at least three biological replicates or cell lines. (E) As for (C), but including cells expressing hp-CAT-B-ACT. doi:10.1371/journal.pone.0025112.g006 increased 2.4-fold by UPF1 RNAi (Figure S5 A,B, average of two independent measurements). The origin of the 4.5 kb band is not clear (see Figure S5, legend); it may be a dicistronic transcript. We also examined the EP procyclin locus. This produces two RNAs which should be targeted by classical NMD, and one of these accumulated 1.3–1.5-fold upon UPF1 depletion (Figure S5C, D). Thus although UPF1 might play some role in control of aberrantly processed mRNAs, the only effects detected were marginal. Discussion This paper includes some results that support the existence of a classical NMD pathway in T. brucei. There are genes encoding UPF1 and UPF2, and the two proteins interact; also, tethering of UPF1 to an mRNA decreased its abundance and stability. The abundances of an endogenous mRNA and a reporter mRNA were decreased by introduction of a PTC. In a reporter mRNA with a full-length CAT ORF, extension of the 39-untranslated region caused a decrease in reporter mRNA abundance which was abrogated by RNAi targeting UPF1. Many other results, however, do not support the existence of classical NMD – notably, several failures to specifically reverse apparent NMD effects by UPF1 RNAi. The decreases in mRNAs upon introduction of PTCs were analogous to those that have been seen in other organisms. For GPI-PLC, a termination codon at codon 17 reduced RNA to 35% of normal, but one at codon 102 only to 80%. For CAT, in contrast, a PTC at codon 81 reduced mRNA to about 35%, and again, early PTCs depressed mRNA levels more than later ones. These values are consistent with NMD in Opisthokonts. For example, PTCs at codon 83 in the Drosophila ADH gene reduced mRNA to 25–30% [64] and mammalian intron-dependent NMD (codon 46) depressed mRNA to 9% [65]. In S. pombe, intron- independent decay reduced mRNA to 43% at codon 27 and 83% at codon 140, while intron-dependent decay resulted in levels around 20% [27]. In Opisthokonts, mechanisms for PTC recognition are predominantly based on (1) the termination codon sequence context; (2) the positions of cis splicing junctions (marked by the EJC); and (3) the length of the 39-UTR (as judged by the distance PLoS ONE | www.plosone.org 9 September 2011 | Volume 6 | Issue 9 | e25112