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Novel Upf2p orthologues suggest a functional link between translation initiation and nonsense surveillance complexes
J T Mendell1, S M Medghalchi, R G Lake
1Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Abstract:
Transcripts harboring premature signals for translation termination are recognized and rapidly degraded by eukaryotic cells through a pathway known as nonsense-mediated mRNA decay (NMD). In addition to protecting cells by preventing the translation of potentially deleterious truncated peptides, studies have suggested that NMD plays a broader role in the regulation of the steady-state levels of physiologic transcripts. In Saccharomyces cerevisiae, three trans-acting factors (Upf1p to Upf3p) are required for NMD. Orthologues of Upf1p have been identified in numerous species, showing that the NMD machinery, at least in part, is conserved through evolution. In this study, we demonstrate additional functional conservation of the NMD pathway through the identification of Upf2p homologues in Schizosaccharomyces pombe and humans (rent2). Disruption of S. pombe UPF2 established that this gene is required for NMD in fission yeast. rent2 was demonstrated to interact directly with rent1, a known trans-effector of NMD in mammalian cells. Additionally, fragments of rent2 were shown to possess nuclear targeting activity, although the native protein localizes to the cytoplasmic compartment. Finally, novel functional domains of Upf2p and rent2 with homology to eukaryotic initiation factor 4G (eIF4G) and other translational regulatory proteins were identified. Directed mutations within these so-called eIF4G homology (4GH) domains were sufficient to abolish the function of S. pombe Upf2p. Furthermore, using the two-hybrid system, we obtained evidence for direct interaction between rent2 and human eIF4AI and Sui1, both components of the translation initiation complex. Based on these findings, a novel model in which Upf2p and rent2 effects decreased translation and accelerated decay of nonsense transcripts through competitive interactions with eIF4G-binding partners is proposed.
Insights
Nonsense-mediated mRNA decay (NMD) degrades faulty transcripts. This study identifies conserved NMD factors, Upf2p and rent2, revealing their role in regulating gene expression and protein translation through interactions with translation initiation factors.
Area of Science:
- Molecular Biology
- Genetics
- Cell Biology
Background:
- Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that degrades aberrant transcripts with premature termination codons.
- NMD prevents the synthesis of truncated, potentially harmful proteins and regulates the expression of normal genes.
- Key NMD factors, like Upf1p, are conserved across species, suggesting a conserved NMD mechanism.
Purpose of the Study:
- To investigate the functional conservation of the NMD pathway by identifying and characterizing Upf2p homologues in Schizosaccharomyces pombe and humans.
- To elucidate the molecular mechanisms by which Upf2p and its human homologue, rent2, contribute to NMD.
- To propose a novel model for NMD regulation involving interactions with translation initiation factors.
Main Methods:
- Identification of Upf2p homologues in S. pombe and humans (rent2).
- Functional analysis of UPF2 in S. pombe by gene disruption.
- Protein interaction studies, including two-hybrid assays and co-immunoprecipitation, to assess interactions between rent2, rent1, eIF4AI, and Sui1.
- Analysis of protein domains and subcellular localization.
Main Results:
- Upf2p homologues were identified in S. pombe and humans (rent2), confirming functional conservation of the NMD pathway.
- Disruption of S. pombe UPF2 demonstrated its essential role in NMD.
- rent2 directly interacts with the known NMD factor rent1 and with components of the translation initiation complex (eIF4AI, Sui1).
- Novel domains with homology to eukaryotic initiation factor 4G (eIF4G) were identified in Upf2p and rent2, and mutations in these domains abolished S. pombe Upf2p function.
Conclusions:
- The NMD pathway, including Upf2p and rent2, is functionally conserved across eukaryotes.
- Upf2p and rent2 play critical roles in NMD by interacting with translation initiation factors.
- A novel model is proposed where Upf2p and rent2 modulate translation and decay of aberrant transcripts through competitive interactions with eIF4G-binding partners.