Is There a Classical Nonsense-Mediated Decay Pathway in Trypanosomes? Praveen Delhi1*, Rafael Queiroz1, Diana Inchaustegui1, Mark Carrington 2 , Christine Clayton 1 * 1 Zentrum f u¨r Molekulare Biologie der Universita¨t Heidelberg, DKFZ-ZMBH Alliance, Hedielberg, Germany, 2 Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom Abstract In many eukaryotes, messenger RNAs with premature termination codons are destroyed by a process called ‘‘nonsense- mediated decay’’, which requires the RNA helicase Upf1 and also, usually, an interacting factor, Upf2. Recognition of premature termination codons may rely on their distance from either a splice site or the polyadenylation site, and long 39- untranslated regions can trigger mRNA decay. The protist Trypanosoma brucei relies heavily on mRNA degradation to determine mRNA levels, and 39-untranslated regions play a major role in control of mRNA decay. We show here that trypanosomes have a homologue of Upf1, TbUPF1, which interacts with TbUPF2 and (in an RNA-dependent fashion) with poly(A) binding protein 1, PABP1. Introduction of a premature termination codon in either an endogenous gene or a reporter gene decreased mRNA abundance, as expected for nonsense-mediated decay, but a dependence of this effect on TbUPF1 could not be demonstrated, and depletion of TbUPF1 by over 95% had no effect on parasite growth or the mRNA transcriptome. Further investigations of the reporter mRNA revealed that increases in open reading frame length tended to increase mRNA abundance. In contrast, inhibition of translation, either using 59-secondary structures or by lengthening the 59-untranslated region, usually decreased reporter mRNA abundance. Meanwhile, changing the length of the 39- untranslated region had no consistent effect on mRNA abundance. We suggest that in trypanosomes, translation per se may inhibit mRNA decay, and interactions with multiple RNA-binding proteins preclude degradation based on 39-untranslated region length alone. Citation: Delhi P, Queiroz R, Inchaustegui D, Carrington M, Clayton C (2011) Is There a Classical Nonsense-Mediated Decay Pathway in Trypanosomes? PLoS ONE 6(9): e25112. doi:10.1371/journal.pone.0025112 Editor: Najib M. El-Sayed, The University of Maryland, United States of America Received July 7, 2011; Accepted August 24, 2011; Published September 21, 2011 Copyright: 2011 Delhi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by the Land of Baden Wu¨ rttemberg (core funding to CC), by a Wellcome Trust grant to CC and MC, and by the Deutsche Forschunggemeinshaft (TM). Rafael Queiroz was supported by a fellowship from the Deutsches Akademisches Austauschdienst. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: pdelhi3@googlemail.com (PD); cclayton@zmbh.uni-heidelberg.de (CC) Introduction The eukaryotic nonsense mediated decay (NMD) pathway degrades mRNAs with mutations that result in premature termination of translation [1,2,3]. Premature termination codons (PTC) can occur through frame-shift or point mutations, or as a consequence of splicing defects. NMD requires translation in order to recognise a PTC, and occurs in the cytoplasm [4,5]. An ATP- dependent RNA helicase called Upf1 is essential for NMD [6,7,8,9,10]. The helicase activity is required [11]: over-expression of a helicase-dead Upf1 mutant had a dominant-negative effect [12]. Some forms of NMD also require Upf2, which interacts with Upf1. Upf1 sequences have been found in all eukaryotic groups tested so far [13,14]. In contrast some organisms have no obvious Upf2, and its loss is correlated with mutations in the Upf2- interaction domain of Upf1 [14,15]. NMD in mammals involves phosphorylation of Upf1 by the Smg-1 kinase; it is not clear if the phosphorylation is necessary in yeast and Smg-1 is not conserved [16]. A nonsense codon can be recognised as a PTC by various mechanisms, depending on both the gene and the species. In several organisms, the nature of the 39-UTR is important: NMD can be triggered by the presence of an abnormally long 39-UTR, or by specific sequences in the 39-UTR or around the termination codon [17]. In a recent study of human cells, Upf1 loading on several mRNAs was shown to be directly proportional to UTR length, suggesting that UPF1 was able to bind non-specifically to the parts of the mRNA that were not being actively translated and thereby ‘‘measure’’ the 39-UTR length [18]. Most Saccharomyces cerevisiae 39UTRs are less than 300 bases long [19] and the presence of a 39-UTR that is longer than usual can trigger NMD [20]. Similarly, the distance from the PTC to the poly(A) tail has been shown to determine NMD in Drosophila melanogaster [21,22], and long 39-UTRs are associated with NMD in Arabidopsis thaliana [23]. This type of NMD depends on ribosome release factors, which can complex either with Upf1 or with poly(A) binding protein (PABP). A current model suggests that when a 39-UTR is abnormally long, interactions with Upf1 predominate, resulting in recruitment of the mRNA decay machinery [15,21,24,25]. Upf2 is not obligatory for this type of NMD in yeast [26] or human cells [15]. In S. pombe the determinants for NMD are uncertain, the main criterion appearing to be the ORF length [27]. Giardia, too, exhibits NMD-like mRNA decay, but the precise signals are unknown and dependence on UPF1 has not been demonstrated [13]. Mammalian cells have a splicing-dependent NMD pathway, which depends on the proteins of the exon junction complex (EJC), which are deposited 220 to 224 nucleotides upstream of PLoS ONE | www.plosone.org 1 September 2011 | Volume 6 | Issue 9 | e25112