Incomplete nonsense-mediated mRNA decay in Giardia lamblia.
Messenger RNAs containing premature translation stop codons are degraded by a nonsense-mediated mRNA decay (NMD) system. The NMD pathway is present in yeast, plants and mammals and is thought to protect cells from production of nonfunctional proteins by rapidly degrading mutant mRNAs. There is little understanding of the biology of the origins of eukaryotes, particularly of the NMD pathway. Searches using the BLAST program revealed that the protozoan Giardialamblia has only some of the components of the NMD pathway. We developed a luciferase reporter system with a nonsense mutation to monitor NMD in Giardia. The nonsense mutation triggered a decrease in luciferase mRNA levels and stability, suggesting that the NMD phenomenon could be present in Giardia. We also found a significant reduction of the mRNA levels of another system containing Giardia its own cyst wall protein 3 gene with a nonsense mutation. However, the reduction levels observed in these two systems are lower than that in late-branching eukaryotes, suggesting that the NMD system in Giardia may be less functional. Interestingly, the effect of G418 in promoting read-through of the nonsense mutation and inhibiting NMD in Giardia is similar to that in late-branching eukaryotes. We also characterised the giardial homologue of a conserved NMD factor, UPF1. Immunofluorescence assays revealed that giardial UPF1, like yeast UPF1, is expressed in the cytoplasm, but not in the nucleus. In addition, overexpression of UPF1 resulted in a reduction of the levels of nonsense-containing transcripts and enhanced translation termination at a nonsense codon. These results suggest that Giardia may have an incomplete NMD pathway and giardial UPF1 may be functionally conserved, involved in NMD and in preventing nonsense suppression.
Chen
Yi-Hsiu
YH
Department of Parasitology, College of Medicine, National Taiwan University, Taipei, Taiwan, ROC.
Su
Li-Hsin
LH
Sun
Chin-Hung
CH
D000818:Animals; D018389:Codon, Nonsense / Q000235:genetics / Q000378:metabolism*; D039601:Eukaryotic Initiation Factor-4A / Q000235:genetics / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D016829:Giardia lamblia / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D020871:RNA Stability / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016053:RNA, Protozoan / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D014162:Transfection / Q000379:methods; D053058:Trophozoites / Q000378:metabolism
D018389:Codon, Nonsense; D012333:RNA, Messenger; D016053:RNA, Protozoan; D016601:RNA-Binding Proteins; D039601:Eukaryotic Initiation Factor-4A