Upf1p, Nmd2p, and Upf3p are interacting components of the yeast nonsense-mediated mRNA decay pathway.

Rapid turnover of nonsense-containing mRNAs in Saccharomyces cerevisiae is dependent on Upf1p, Nmd2p, and Upf3p, the products of the UPF1, NMD2/UPF2, and UPF3 genes, respectively. We showed previously that Upf1p and Nmd2p interact and that this interaction is required for nonsense-mediated mRNA decay (F. He and A. Jacobson, Genes Dev. 9:437-454, 1995; F. He, A. H. Brown, and A. Jacobson, RNA 2:153-170, 1996). In this study we have used the yeast two-hybrid system to define other protein-protein interactions among the essential components of this decay pathway. Nmd2p-Upf3p and Upf1p-Upf3p interactions were identified, and the respective domains involved in these interactions were delineated by deletion analysis. The domains of Upf1p and Upf3p putatively involved in their mutual interaction were found to correspond to the domains on the two proteins which interact with Nmd2p, suggesting that Nmd2p bridges Upf1p and Upf3p. This conclusion was reinforced by experiments showing that: (i) deletion of NMD2 completely abolishes interactions between Upf1p and Upf3p and (ii) overexpression of full-length Nmd2p or Nmd2p fragments that retain Upf1p- and Upf3p-interacting domains promotes 10- to 200-fold enhancement of Upf1p-Nmd2p-Upf3p complex formation. These results; the observation that cells harboring either single or multiple deletions of UPF1, NMD2, and UPF3 inhibit nonsense-mediated mRNA decay to the same extent; and an analysis of the possible targets of a dominant-negative NMD2 allele indicate that Upf1p, Nmd2p, Upf3p, and at least one other factor are functionally dependent, interacting components of the yeast nonsense-mediated mRNA decay pathway.     

Role for Upf2p phosphorylation in Saccharomyces cerevisiae nonsense-mediated mRNA decay

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.     

CBP80 promotes interaction of Upf1 with Upf2 during nonsense-mediated mRNA decay in mammalian cells

In mammalian cells, nonsense-mediated messenger RNA decay (NMD) targets newly synthesized nonsense-containing mRNA bound by the cap-binding-protein heterodimer CBP80-CBP20 and at least one exon-junction complex (EJC). An EJC includes the NMD factors Upf3 or Upf3X and Upf2, and Upf2 recruits Upf1. Once this pioneer translation initiation complex is remodeled so that CBP80-CBP20 is replaced by eukaryotic initiation factor 4E, the mRNA is no longer detectably targeted for NMD. Here, we provide evidence that CBP80 augments the efficiency of NMD but not of Staufen1 (Stau1)-mediated mRNA decay (SMD). SMD depends on the recruitment of Upf1 by the RNA-binding protein Stau1 but does not depend on the other Upf proteins. We find that CBP80 interacts with Upf1 and promotes the interaction of Upf1 with Upf2 but not with Stau1.     

Interaction between Nmd2p and Upf1p is required for activity but not for dominant-negative inhibition of the nonsense-mediated mRNA decay pathway in yeast.

Rapid turnover of nonsense-containing mRNAs in the yeast Saccharomyces cerevisiae is dependent on the products of the UPF1 (Upf1p), NMD2/UPF2 (Nmd2p) and UPF3 (Upf3p) genes. Mutations in each of these genes lead to the selective stabilization of mRNAs containing early nonsense mutations without affecting the decay rates of most other mRNAs. NMD2 was recently identified in a two-hybrid screen as a gene that encodes a Upf1p-interacting protein. To identify the amino acids essential to this interaction, we used two-hybrid analysis as well as missense, nonsense, and deletion mutants of NMD2, and mapped the Upf1p-interacting domain of Nmd2p to a 157-amino acid segment at its C-terminus. Mutations in this domain that disrupt interaction with Upf1p also disrupt nonsense-mediated mRNA decay. A dominant-negative deletion allele of NMD2 identified previously includes the Upf1p-interacting domain. However, mutations in the Upf1p-interacting domain do not affect dominant-negative inhibition of mRNA decay caused by this allele, suggesting interaction with yet another factor. These results, and the observation that deletion of a putative nuclear localization signal and a putative transmembrane domain also inactivate nonsense-mediated mRNA decay, suggest that Nmd2p may contain as many as four important functional domains.     

Human Upf proteins target an mRNA for nonsense-mediated decay when bound downstream of a termination codon

Nonsense-mediated decay (NMD) rids eukaryotic cells of aberrant mRNAs containing premature termination codons. These are discriminated from true termination codons by downstream cis-elements, such as exon-exon junctions. We describe three novel human proteins involved in NMD, hUpf2, hUpf3a, and hUpf3b. While in HeLa cell extracts these proteins are complexed with hUpf1, in intact cells hUpf3a and hUpf3b are nucleocytoplasmic shuttling proteins, hUpf2 is perinuclear, and hUpf1 cytoplasmic. hUpf3a and hUpf3b associate selectively with spliced beta-globin mRNA in vivo, and tethering of any hUpf protein to the 3'UTR of beta-globin mRNA elicits NMD. These data suggest that assembly of a dynamic hUpf complex initiates in the nucleus at mRNA exon-exon junctions and triggers NMD in the cytoplasm when recognized downstream of a translation termination site.     

Nuclear import of Upf3p is mediated by importin-alpha/-beta and export to the cytoplasm is required for a functional nonsense-mediated mRNA decay pathway in yeast

Upf3p, which is required for nonsense-mediated mRNA decay (NMD) in yeast, is primarily cytoplasmic but accumulates inside the nucleus when UPF3 is overexpressed or when upf3 mutations prevent nuclear export. Upf3p physically interacts with Srp1p (importin-alpha). Upf3p fails to be imported into the nucleus in a temperature-sensitive srp1-31 strain, indicating that nuclear import is mediated by the importin-alpha/beta heterodimer. Nuclear export of Upf3p is mediated by a leucine-rich nuclear export sequence (NES-A), but export is not dependent on the Crm1p exportin. Mutations identified in NES-A prevent nuclear export and confer an Nmd(-) phenotype. The addition of a functional NES element to an export-defective upf(-) allele restores export and partially restores an Nmd(+) phenotype. Our findings support a model in which the movement of Upf3p between the nucleus and the cytoplasm is required for a fully functional NMD pathway. We also found that overexpression of Upf2p suppresses the Nmd(-) phenotype in mutant strains carrying nes-A alleles but has no effect on the localization of Upf3p. To explain these results, we suggest that the mutations in NES-A that impair nuclear export cause additional defects in the function of Upf3p that are not rectified by restoration of export alone.     

Arabidopsis UPF1 RNA helicase for nonsense-mediated mRNA decay is involved in seed size control and is essential for growth

UPF1 RNA helicase plays a central role in nonsense-mediated mRNA decay (NMD), which specifically recognizes aberrant mRNAs containing premature termination codons and targets them for degradation. Although NMD factors are highly conserved among eukaryotes, little is known about the role of NMD in plant growth and development. The lba1 mutant of Arabidopsis thaliana with a Gly(851)-->Glu missense mutation in AtUPF1 yielded seeds that were on average 22% longer in the long axis and 35% heavier than the wild-type Col seeds. Expression of the wild-type AtUPF1 in this mutant reduced the seeds to a normal size. During early stages of seed development, globular to torpedo stages of the embryos were contained within seed sacs that were larger in lba1 than in Col. Furthermore, the distance between seeds in siliques was greater in lba1 than in Col, suggesting that the lba1 mutation may affect ovule development. Self-pollinated atupf1-3(+/-) plants heterozygous for AtUPF1 disrupted by T-DNA insertion developed atupf1-3(-/-) seeds with a size and shape similar to those of Col seeds. However, the atupf1-3(-/-) seedlings stopped growing after radicle emergence from the seed coat, and this seedling lethality was rescued by expressing the wild-type AtUPF1. These results suggest that the lba1 mutation in AtUPF1 maternally affects seed development and that AtUPF1 is essential for seedling growth.     

The serine/threonine kinase Cmk2 is required for oxidative stress response in fission yeast

Cmk2, a fission yeast Ser/Thr protein kinase homologous to mammalian calmodulin kinases, is essential for oxidative stress response. Cells lacking cmk2 gene were specifically sensitive to oxidative stress conditions. Upon stress, Cmk2 was phosphorylated in vivo, and this phosphorylation was dependent on the stress-activated MAPK Sty1/Spc1. Co-precipitation assays demonstrated that Cmk2 binds Sty1. Furthermore, in vivo or in vitro activated Sty1 was able to phosphorylate Cmk2, and the phosphorylation occurred at the C-terminal regulatory domain at Thr-411. Cell lethality caused by overexpression of Wis1 MAPK kinase was abolished by deletion of cmk2 or by mutation of Thr-411 of Cmk2. Taken together, our data suggest that Cmk2 acts downstream of Sty1 and is an essential kinase for oxidative stress responses.     

Upf1p, a highly conserved protein required for nonsense-mediated mRNA decay, interacts with the nuclear pore proteins Nup100p and Nup116p

Saccharomyces cerevisiae Upf1p is a 971-amino-acid protein that is required for the nonsense-mediated mRNA decay (NMD) pathway, a pathway that degrades mRNAs with premature translational termination codons. We have identified a two-hybrid interaction between Upf1p and the nuclear pore (Nup) proteins, Nup100p and Nup116p. Both nucleoporins predominantly localize to the cytoplasmic side of the nuclear pore and participate in mRNA transport. The two-hybrid interaction between Upf1p and the nuclear pore proteins, Nup100p and Nup116p, is dependent on the presence of the C-terminal 158 amino acids of Upf1p. Nup100p and Nup116p can be co-immunoprecipitated from whole-cell extracts with Upf1p, confirming in vitro the interaction identified by the two-hybrid analysis. Finally, we see a genetic interaction between UPF1 and NUP100. The growth of upf1Delta, can1-100 cells is inhibited by canavanine. The deletion of NUP100 allows upf1Delta, can1-100 cells to grow in the presence of canavanine. Physiologically, the interaction between Upf1p and the nuclear pore proteins, Nup100p and Nup116p, is significant because it suggests a mechanism to ensure that Upf1p associates with newly synthesized mRNA as it is transported from the nucleus to the cytoplasm prior to the pioneer round of translation.     

Characterization of the biochemical properties of the human Upf1 gene product that is involved in nonsense-mediated mRNA decay

The Upf1 protein in yeast has been implicated in the modulation of efficient translation termination as well as in the accelerated turnover of mRNAs containing premature stop codons, a phenomenon called nonsense-mediated mRNA decay (NMD). A human homolog of the yeast UPF1, termed HUpf1/RENT1, has also been identified. The HUpf1 has also been shown to play a role in NMD in mammalian cells. Comparison of the yeast and human UPF1 proteins demonstrated that the amino terminal cysteine/histidine-rich region and the region comprising the domains that define this protein as a superfamily group I helicase have been conserved. The yeast Upf1p demonstrates RNA-dependent ATPase and 5' --> 3' helicase activities. In this paper, we report the expression, purification, and characterization of the activities of the human Upf1 protein. We demonstrate that human Upf1 protein displays a nucleic-acid-dependent ATPase activity and a 5'--> 3' helicase activity. Furthermore, human Upf1 is an RNA-binding protein whose RNA-binding activity is modulated by ATP. Taken together, these results indicate that the activities of the Upf1 protein are conserved across species, reflecting the conservation of function of this protein throughout evolution.     

A role for AKT1 in nonsense-mediated mRNA decay

Nonsense-mediated mRNA decay (NMD) is a highly regulated quality control mechanism through which mRNAs harboring a premature termination codon are degraded. It is also a regulatory pathway for some genes. This mechanism is subject to various levels of regulation, including phosphorylation. To date only one kinase, SMG1, has been described to participate in NMD, by targeting the central NMD factor UPF1. Here, screening of a kinase inhibitor library revealed as putative NMD inhibitors several molecules targeting the protein kinase AKT1. We present evidence demonstrating that AKT1, a central player in the PI3K/AKT/mTOR signaling pathway, plays an essential role in NMD, being recruited by the UPF3X protein to phosphorylate UPF1. As AKT1 is often overactivated in cancer cells and as this should result in increased NMD efficiency, the possibility that this increase might affect cancer processes and be targeted in cancer therapy is discussed.