Human Upf1 protein (p), a group 1 RNA helicase, has recently been shown to function in nonsense-mediated mRNA decay (NMD) in mammalian cells. Here, we demonstrate that the estimated 3 × 106 copies of hUpf1p per exponentially growing HeLa cell are essentially equally distributed among polysomal, subpolysomal, and ribosome-free fractions. We also demonstrate that hUpf1p binds RNA and is a phosphoprotein harboring phosphoserine and phosphothreonine. hUpf1p is phosphorylated to the highest extent when polysome-associated and to the lowest extent when ribosome free. We find that serum-induced phosphorylation of hUpf1p is inhibited by wortmannin at a concentration that selectively inhibits PI 3-kinase related kinases and, to a lesser extent, by rapamycin. These and other data suggest that phosphorylation is mediated by a wortmannin-sensitive and rapamycin-sensitive PI 3-kinase-related kinase signaling pathway. Comparisons are made of hUpf1p to Upf1p and SMG-2, which are the orthologs to hUpf1p in Saccharomyces cerevisiae and Caenorhabditis elegans, respectively.

Translation termination is the final step that completes the synthesis of a polypeptide. Premature translation termination by introduction of a nonsense mutation leads to the synthesis of a truncated protein. We report the identification and characterization of the product of the MTT1 gene, a helicase belonging to the Upf1-like family of helicases that is involved in modulating translation termination. MTT1 is homologous to UPF1, a factor previously shown to function in both mRNA turnover and translation termination. Overexpression of MTT1 induced a nonsense suppression phenotype in a wild-type yeast strain. Nonsense suppression is apparently not due to induction of [PSI+], even though cooverexpression of HSP104 alleviated the nonsense suppression phenotype observed in cells overexpressing MTT1, suggesting a more direct role of Hsp104p in the translation termination process. The MTT1 gene product was shown to interact with translation termination factors and is localized to polysomes. Taken together, these results indicate that at least two members of a family of RNA helicases modulate translation termination efficiency in cells.

In mammals, as in all organisms examined, mRNAs that prematurely terminate translation are abnormally reduced in abundance by a mechanism called nonsense-mediated mRNA decay (NMD) or mRNA surveillance (for reviews, see Maquat, 1995, 1996; Ruiz-Echevarria & Peltz, 1996; Li & Wilkinson, 1998; Culbertson, 1999; Hentze & Kulozik, 1999; Hilleren & Parker, 1999). This mechanism is thought to have evolved to eliminate nonsense-containing RNAs that arise as a consequence of (1) mutations in germ-line or somatic DNA or (2) routine abnormalities in gene expression due to abnormalities in, for example, transcription initiation, splicing, and somatic rearrangements of the type that characterize the immunoglobulin and T-cell receptor genes. The elimination of nonsense-containing mRNAs protects cells from the potentially deleterious effects of the encoded truncated proteins, which can manifest new or dominant-negative functions (Kazazian et al., 1992; Pulak & Anderson, 1993; Hall & Thein, 1994; Cali & Anderson, 1998). In addition to eliminating abnormal transcripts, NMD also regulates the expression of certain mRNAs that are not abnormal. Examples in mammalian cells are provided by certain selenoprotein mRNAs that terminate translation at a UGA codon for the inefficiently incorporated amino acid selenocysteine (Sec; Moriarty et al., 1997, 1998). Other examples will undoubtedly resemble natural substrates found in other organisms such as the alternatively spliced mRNAs of Caenorhabditis elegans that retain an internal exon harboring an in-frame termination codon (Morrison et al., 1997), and transcripts of Saccharomyces cerevisiae that contain a small open reading frame upstream of the primary open reading frame (Leeds et al., 1992; Pierrat et al., 1993).

The yeast NMD3 gene was identified in a two-hybrid screen using the nonsense-mediated mRNA decay factor, Upf1p, as bait. NMD3 was shown to encode an essential, highly conserved protein that associated principally with free 60S ribosomal subunits. Overexpression of a truncated form of Nmd3p, lacking 100 C-terminal amino acids and most of its Upf1p-interacting domain, had dominant-negative effects on both cell growth and protein synthesis and promoted the formation of polyribosome half-mers. These effects were eliminated by truncation of an additional 100 amino acids from Nmd3p. Overexpression of the nmd3Δ100 allele also led to increased synthesis and destabilization of some ribosomal protein mRNAs, and increased synthesis and altered processing of 35S pre-rRNA. Our data suggest that Nmd3p has a role in the formation, function, or maintenance of the 60S ribosomal subunit and may provide a link for Upf1p to 80S monosomes.

The nonsense-mediated mRNA decay pathway decreases the abundance of mRNAs that contain premature termination codons and prevents suppression of nonsense alleles. The UPF1 gene in the yeast Saccharomyces cerevisiae was shown to be a trans-acting factor in this decay pathway. The Upf1p demonstrates RNA-dependent ATPase, RNA helicase, and RNA binding activities. The results presented here investigate the binding affinity of the Upf1p for ATP and the consequences of ATP binding on its affinity for RNA. The results demonstrate that the Upf1p binds ATP in the absence of RNA. Consistent with this result, the TR800AA mutant form of the Upf1p still bound ATP, although it does not bind RNA. ATP binding also modulates the affinity of Upf1p for RNA. The RNA binding activity of the DE572AA mutant form of the Upf1p, which lacks ATPase activity, still bound ATP as efficiently as the wild-type Upf1p and destabilized the Upf1p–RNA complex. Similarly, ATPγS, a nonhydrolyzable analogue of ATP, interacted with Upf1p and promoted disassociation of the Upf1p–RNA complex. The conserved lysine residue (K436) in the helicase motif Ia in the Upf1p was shown to be critical for ATP binding. Taken together, these findings formally prove that ATP can bind Upf1p in the absence of RNA and that this interaction has consequences on the formation of the Upf1p–RNA complex. Further, the results support the genetic evidence indicating that ATP binding is important for the Upf1p to increase the translation termination efficiency at a nonsense codon. Based on these findings, a model describing how the Upf1p functions in modulating translation and turnover and the potential insights into the mechanism of the Upf1p helicase will be discussed.