Is There a Classical Nonsense-Mediated Decay Pathway in Trypanosomes?

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 3′-untranslated regions can trigger mRNA decay. The protist Trypanosoma brucei relies heavily on mRNA degradation to determine mRNA levels, and 3′-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 5′-secondary structures or by lengthening the 5′-untranslated region, usually decreased reporter mRNA abundance. Meanwhile, changing the length of the 3′-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 3′-untranslated region length alone.

Depletion of the Trypanosome Pumilio Domain Protein PUF2 or of Some Other Essential Proteins Causes Transcriptome Changes Related to Coding Region Length

“…To do this, we used a bloodstream-form cell line that expresses a CAT mRNA bearing 5 copies of the boxB recognition sequence. We also inducibly expressed PUF2 with the N peptide at the N terminus and a myc tag on its C terminus (40). The N peptide binds to the boxB sequence and therefore "tethers" any protein to which it is fused to the mRNA (61,62) (Fig.…”

“…For tethering, the PUF2 ORF was PCR amplified using the primers CZ3913 (ApaI) and CZ4025 (BamHI) and ligated into vector pHD2202 (40) to give pHD2304. The plasmid was cotransfected for inducible expression of the fusion protein, N-PUF2-myc, into cell lines constitutively expressing a chloramphenicol acetyltransferase (CAT) reporter with an actin 3=-UTR (pHD1991) and box B actin 3=-UTR (pHD2277).…”

The Pumilio-domain protein PUF6 contributes to SIDER2 retroposon-mediated mRNA decay in Leishmania

“…Development of an improved MS2 coat protein tethering system for identifying trans-acting factors regulating SIDER2 retroposon-mediated mRNA decay in Leishmania So far, the λN peptide has been used successfully to tether RBPs to a particular RNA of interest in the related Trypanosoma species (Delhi et al 2011;Wurst et al 2012;Droll et al 2013;Jha et al 2014;Singh et al 2014). Here, we have developed an improved MS2 coat protein tethering system adapted for use in Leishmania to attach RNA-binding proteins (RBPs) specifically to a reporter RNA.…”

The evolution and diversity of the nonsense-mediated mRNA decay pathway

“…The excavata have been suggested to be the most basal group of eukaryotes 37 , although other work places them within the same supergroup as plants 38, 39 . Although the NMD pathways of the parasites Giardia lamblia and Trypanosoma brucei have been studied, it is unclear if a functional NMD pathway exists in these organisms 40, 41 . They contain heavily reduced compliments of NMD factors: the genome of G. lamblia only harbors UPF1, and the genome of T. brucei only harbors UPF1 and UPF2 40, 41 .…”

“…Although the NMD pathways of the parasites Giardia lamblia and Trypanosoma brucei have been studied, it is unclear if a functional NMD pathway exists in these organisms 40, 41 . They contain heavily reduced compliments of NMD factors: the genome of G. lamblia only harbors UPF1, and the genome of T. brucei only harbors UPF1 and UPF2 40, 41 . Over-expression of UPF1 in G. lamblia caused an NMD reporter to further decrease, suggesting that G. lamblia might have an active NMD pathway 40 .…”