S4. Expression of lambdaN-UPF1-flag was induced with tetracy- cline. In the absence of lambdaN-UPF1-FLAG induction, the half- life of the CAT-B-ACT mRNA was 15–20 min. This was surprisingly short. We do not understand why, but one possible explanation is that the mRNAs were made by T7 polymerase. Instability of a T7-produced CAT reporter with a different 39- UTR has been previously observed; the reasons are unknown (Colasante et al., Mol Biochem Parasitol 151, 193–204.) Upon induction of lambdaN-UPF1-FLAG expression the half-life decreased further. (PDF) Figure S5 RNAi targeting TbUPF1 has minor effects on the abundances of mRNAs from two loci. (A) shows a map of the region around Tb927.10.12900. The direction of transcrip- tion and the positions of various probes (a–d, dotted lines) used to identify the various RNAs is indicated. Estimated sizes and location of monocistronic RNAs, based on our own mapping and on RNASeq tag abundances shown at tritrypDB, are also shown. (B) Shows a Northern blot of RNA with and without RNAi targeting either XRNA or UPF1, using probe (a) (made using CZ3585, CZ3586). A Histone H4 probe (HISH4) serves as a control. Probe ‘c’ (made using oligonucleotides CZ3695, CZ3696) detected a single RNA migrating at 3.7 kb and probe ‘d’ (made using CZ3707, CZ3708) detected two RNAs of similar abundance, migrating at 2.6 kb and 1.8 kb. The band of 4.5 kb was detected only by probes (a) and (b). There is a tag gap in the middle of Tb10.389.0620, but no mapped spliced leader addition site. (C) The procyclin locus containing EP1 and EP2, with predicted transcripts based on mapped splicing sites. Alternative splicing of the procyclin EP2 RNA precursor is known to result in two transcripts. The dominant one, at 0.9 kb, has a short 59-UTR preceding the EP2 initiation codon. The minor one, of 1.4 kb, has various very short ORFs upstream of the EP2 ORF while the 0.4 kb RNA contains these short ORFs alone: either of these might be a substrate for NMD. The location of the EP2 upstream probe used in (D) is indicated as a dotted line. (D) RNA was References 1. Chang Y-F, Imam J, Wilkinson M (2007) The Nonsense-Mediated Decay RNA surveillance pathway. Ann Rev Biochem 76: 51–74. 2. Maquat LE (2005) Nonsense-mediated mRNA decay in mammals. J Cell Sci 118: 1773–1776. 3. Stalder L, Mu¨ hlemann O (2008) The meaning of nonsense. Trends Cell Biol 18: 315–321. 4. Kuperwasser N, Brogna S, Dower K, Rosbash M (2004) Nonsense-mediated decay does not occur within the yeast nucleus. Rna 10: 1907–1915. 5. Singh G, Jakob S, Kleedehn M, Lykke-Andersen J (2007) Communication with the exon-junction complex and activation of nonsense-mediated decay by human Upf proteins occur in the cytoplasm. Molecualr Cell 27: 780–792. 6. 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Weng Y, Czaplinski K, Peltz S (1996) Genetic and biochemical characterization of mutations in the ATPase and helicase regions of the Upf1 protein. Mol Cell Biol 16: 5477–5490. 12. Sun X, Perlick H, Dietz H, Maquat L (1998) A mutated human homologue to yeast Upf1 protein has a dominant-negative effect on the decay of nonsense-containing mRNAs in mammalian cells. Proc Natl Acad Sci U S A 95: 10009–10014. Nonsense-Mediated Decay in Trypanosomes prepared from three independent bloodstream-form trypanosome clones (AnTat1.1. strain) with TbUPF1 hpRNAi, incubated with 100 ng/ml tetracycline for 2 days. The blot was probed with the EP2 upstream region indicated in (A). We detected the 0.9 kb EP mRNAs and three additional bands at 0.4, 1.4 and 2.3 kb. The 2.3 kb signal comigrates with rRNA so could either be a cross- hybridisation or a dicistronic precursor. The bands were all of approximately equal intensity: the mature EP mRNA is very unstable in bloodstream forms. 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