Overexpression of Upf1p compensates for mitochondrial splicing deficiency independently of its role in mRNA surveillance

Pinto, B. de; Lippolis, Rosa; Castaldo, Rosa; Altamura, Nicola

doi:10.1046/j.1365-2958.2003.03889.x

Cited by 23 publications

(20 citation statements)

References 49 publications

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“…5), as was reported by another group while the manuscript was in preparation (11). This finding is significant, as the lack of previous evidence for yeast Upf1p phosphorylation had prompted some investigators to suggest that the NMD pathway in S. cerevisiae is controlled by a mechanism independent of Upf1p phosphorylation (12).…”

Section: Discussionsupporting

confidence: 65%

Role for Upf2p Phosphorylation in Saccharomyces cerevisiae Nonsense-Mediated mRNA Decay

Wang¹,

Cajigas

Peltz

et al. 2006

Molecular and Cellular Biology

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Premature termination (nonsense) codons trigger rapid mRNA decay by the nonsense-mediated mRNA decay (NMD) pathway. Two conserved proteins essential for NMD, UPF1 and UPF2, are phosphorylated in higher eukaryotes. The phosphorylation and dephosphorylation of UPF1 appear to be crucial for NMD, as blockade of either event in Caenorhabditis elegans and mammals largely prevents NMD. The universality of this phosphorylation/dephosphorylation cycle pathway has been questioned, however, because the well-studied Saccharomyces cerevisiae NMD pathway has not been shown to be regulated by phosphorylation. Here, we used in vitro and in vivo biochemical techniques to show that both S. cerevisiae Upf1p and Upf2p are phosphoproteins. We provide evidence that the phosphorylation of the N-terminal region of Upf2p is crucial for its interaction with Hrp1p, an RNA-binding protein that we previously showed is essential for NMD. We identify specific amino acids in Upf2p's N-terminal domain, including phosphorylated serines, which dictate both its interaction with Hrp1p and its ability to elicit NMD. Our results indicate that phosphorylation of UPF1 and UPF2 is a conserved event in eukaryotes and for the first time provide evidence that Upf2p phosphorylation is crucial for NMD.Cells have evolved many quality control mechanisms to eliminate aberrant proteins and mRNAs that interfere with normal cellular functions. One such mechanism is the nonsense-mediated mRNA decay (NMD) pathway, which eliminates mRNAs that contain premature termination (nonsense) codons within the protein coding region, thereby preventing the synthesis of truncated proteins with dominant-negative and deleterious gainof-function activities (3,7,14,16,24,42,47). The importance of this surveillance mechanism is underscored by its conservation in a wide variety of organisms, including Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, plants, and mammals (12,13,16,25,27,34,38).Several genes essential for NMD have been identified in yeast, most notably UPF1, UPF2, and UPF3, all of which destabilize nonsense codon-containing mRNAs without affecting the decay rate of most wild-type mRNAs (16,25). The yeast UPF1 gene encodes the protein Upf1p, which has RNA-binding and RNA-dependent ATPase/helicase activities (8, 9, 45). Yeast UPF3 encodes the basic protein Upf3p, which harbors several nuclear localization and nuclear export signals that allow the protein to shuttle between the nucleus and the cytoplasm (39, 40). Yeast UPF2 encodes the adaptor protein Upf2p, which forms a complex with both Upf1p and Upf3p (6, 19). Single or multiple deletions of each of these three UPF genes produce similar effects on mRNA decay, consistent with the notion that the Upf proteins function as a molecular complex in a single pathway (19).The NMD pathway is elicited by recognition of a nonsense codon, but the precise features distinguishing a premature termination codon from a bona fide stop codon remain unknown. In S. cerevisiae, one model for NMD suggests that the de...

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Section: Discussionsupporting

confidence: 65%

Role for Upf2p Phosphorylation in Saccharomyces cerevisiae Nonsense-Mediated mRNA Decay

Wang¹,

Cajigas

Peltz

et al. 2006

Molecular and Cellular Biology

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“…Human and Drosophila orthologs of smg-1, smg-5, smg-6, and smg-7 are known to be involved in NMD and in UPF1/SMG-2 phosphorylation, but yeast orthologs have not been described. Nevertheless, the phosphorylation of Upf1p in yeast cells was recently described (20). Yeast cells do not have an apparent smg-1 ortholog, but they do contain other members of the PIK-related family of kinases.…”

Section: Discussionmentioning

confidence: 99%

SMG-1 Is a Phosphatidylinositol Kinase-Related Protein Kinase Required for Nonsense-Mediated mRNA Decay inCaenorhabditis elegans

Grimson

O’Connor

Newman

et al. 2004

Molecular and Cellular Biology

114

120

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Eukaryotic messenger RNAs containing premature stop codons are selectively and rapidly degraded, a phenomenon termed nonsense-mediated mRNA decay (NMD). Previous studies with both Caenohabditis elegans and mammalian cells indicate that SMG-2/human UPF1, a central regulator of NMD, is phosphorylated in an SMG-1-dependent manner. We report here that smg-1, which is required for NMD in C. elegans, encodes a protein kinase of the phosphatidylinositol kinase superfamily of protein kinases. We identify null alleles of smg-1 and demonstrate that SMG-1 kinase activity is required in vivo for NMD and in vitro for SMG-2 phosphorylation. SMG-1 and SMG-2 coimmunoprecipitate from crude extracts, and this interaction is maintained in smg-3 and smg-4 mutants, both of which are required for SMG-2 phosphorylation in vivo and in vitro. SMG-2 is located diffusely through the cytoplasm, and its location is unaltered in mutants that disrupt the cycle of SMG-2 phosphorylation. We discuss the role of SMG-2 phosphorylation in NMD.Nonsense-mediated mRNA decay (NMD) rapidly and selectively degrades eukaryotic mRNAs containing premature translation termination codons (PTCs) (reviewed in references 27, 43, and 65). NMD likely improves the fidelity of gene expression by degrading aberrant mRNAs, thereby protecting cells from potentially deleterious consequences of their translation (9, 28, 53). The biological sources of NMD substrates are only partially understood. For example, unspliced, unproductively spliced, and aberrantly spliced mRNAs are degraded by NMD (29,39,48). NMD strongly influences the expression of certain genes for which mRNAs containing PTCs are a normal feature of their expression. For example, gene rearrangements of T-cell receptor genes often result in mRNAs that contain PTCs and are subjected to NMD (11).Gene products required for NMD have been identified in fungi, nematodes, insects, and mammals (13,16,24,49), and putative orthologs of these genes are evident in many sequenced eukaryotic genomes. A core group of three genes first identified in the yeast Saccharomyces cerevisiae (UPF1, NMD2/ UPF2, and UPF3) (reviewed in reference 69) are required for NMD in all tested eukaryotes (3,40,45,50,57,61). The conservation of both the sequence and function of such genes indicates that NMD is an evolutionarily ancient process and suggests that elements of the mechanism of NMD will be similar in most, or perhaps all, eukaryotes.PTCs are distinguished from normal termination codons in both yeast and mammalian cells by the context of translation termination. Specific cis-acting elements mark the open reading frame of mRNAs and activate NMD when translation terminates upstream of the marks. The Upf proteins appear to be involved both in sensing the presence of downstream signals and in activating the degradation machinery. In yeast cells, the cis-acting elements are termed downstream sequence elements, which are thought to be present throughout coding sequences but not in 3Ј untranslated regions (54). Hrp1p binds both downstream seq...

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“…A similar model was proposed for S. cerevisiae (Culbertson and Neeno-Eckwall 2005); nevertheless, the association between DSE and Upf3p and/or Upf2p needs to be clarified. Recently, phosphorylation of Upf1p was described in S. cerevisiae (de Pinto et al 2004), although S. cerevisiae lacks an obvious SMG-1 homolog. In addition, Upf1p-C65S, which is a similar mutation to human Upf1-C126S, shows significant phosphorylation, but S. cerevisiae might have different means of Upf1 phosphorylation (de Pinto et al 2004).…”

Section: Smg-1 Phosphorylates Upf1 In Decid After Recognition Of Premmentioning

confidence: 99%

Binding of a novel SMG-1–Upf1–eRF1–eRF3 complex (SURF) to the exon junction complex triggers Upf1 phosphorylation and nonsense-mediated mRNA decay

Kashima¹,

Yamashita²,

Izumi³

et al. 2006

Genes Dev.

552

869

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Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism that degrades mRNA containing premature termination codons (PTCs). In mammalian cells, recognition of PTCs requires translation and depends on the presence on the mRNA with the splicing-dependent exon junction complex (EJC). While it is known that a key event in the triggering of NMD is phosphorylation of the trans-acting factor, Upf1, by SMG-1, the relationship between Upf1 phosphorylation and PTC recognition remains undetermined. Here we show that SMG-1 binds to the mRNA-associated components of the EJC, Upf2, Upf3b, eIF4A3, Magoh, and Y14. Further, we describe a novel complex that contains the NMD factors SMG-1 and Upf1, and the translation termination release factors eRF1 and eRF3 (SURF). Importantly, an association between SURF and the EJC is required for SMG-1-mediated Upf1 phosphorylation and NMD. Thus, the SMG-1-mediated phosphorylation of Upf1 occurs on the association of SURF with EJC, which provides the link between the EJC and recognition of PTCs and triggers NMD.[Keywords: SMG-1; eRF; Upf; phosphorylation; EJC; NMD] Supplemental material is available at http://www.genesdev.org.

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Overexpression of Upf1p compensates for mitochondrial splicing deficiency independently of its role in mRNA surveillance

Cited by 23 publications

References 49 publications

Role for Upf2p Phosphorylation in Saccharomyces cerevisiae Nonsense-Mediated mRNA Decay

Role for Upf2p Phosphorylation in Saccharomyces cerevisiae Nonsense-Mediated mRNA Decay

SMG-1 Is a Phosphatidylinositol Kinase-Related Protein Kinase Required for Nonsense-Mediated mRNA Decay inCaenorhabditis elegans

Binding of a novel SMG-1–Upf1–eRF1–eRF3 complex (SURF) to the exon junction complex triggers Upf1 phosphorylation and nonsense-mediated mRNA decay

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