The role of Upf proteins in modulating the translation read-through of nonsense-containing transcripts.

The yeast UPF1, UPF2 and UPF3 genes encode trans-acting factors of the nonsense-mediated mRNA decay pathway. In addition, the upf1Delta strain demonstrates a nonsense suppression phenotype and Upf1p has been shown to interact with the release factors eRF1 and eRF3. In this report, we show that both upf2Delta and upf3Delta strains demonstrate a nonsense suppression phenotype independent of their effect on mRNA turnover. We also demonstrate that Upf2p and Upf3p interact with eRF3, and that their ability to bind eRF3 correlates with their ability to complement the nonsense suppression phenotype. In vitro experiments demonstrate that Upf2p, Upf3p and eRF1 compete with each other for interacting with eRF3. Con versely, Upf1p binds to a different region of eRF3 and can form a complex with these factors. These results suggest a sequential surveillance complex assembly pathway, which occurs during the premature translation termination process. We propose that the observed nonsense suppression phenotype in the upfDelta strains can be attributed to a defect in the surveillance complex assembly.

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.

Mtt1 is a Upf1-like helicase that interacts with the translation termination factors and whose overexpression can modulate termination efficiency.

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 Upfl-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.

Should we kill the messenger? The role of the surveillance complex in translation termination and mRNA turnover.

Eukaryotes have evolved conserved mechanisms to rid cells of faulty gene products that can interfere with cell function. mRNA surveillance is an example of a pathway that monitors the translation termination process and promotes degradation of transcripts harboring premature translation termination codons. Studies on the mechanism of mRNA surveillance in yeast and humans suggest a common mechanism where a "surveillance complex" monitors the translation process and determines whether translation termination has occurred at the correct position within the mRNA. A model will be presented that suggests that the surveillance complex assesses translation termination by monitoring the transition of an RNP as it is converted from a nuclear to a cytoplasmic form during the initial rounds of translation.

The surveillance complex interacts with the translation release factors to enhance termination and degrade aberrant mRNAs.

The nonsense-mediated mRNA decay pathway is an example of an evolutionarily conserved surveillance pathway that rids the cell of transcripts that contain nonsense mutations. The product of the UPF1 gene is a necessary component of the putative surveillance complex that recognizes and degrades aberrant mRNAs. Recent results indicate that the Upf1p also enhances translation termination at a nonsense codon. The results presented here demonstrate that the yeast and human forms of the Upf1p interact with both eukaryotic translation termination factors eRF1 and eRF3. Consistent with Upf1p interacting with the eRFs, the Upf1p is found in the prion-like aggregates that contain eRF1 and eRF3 observed in yeast [PSI+] strains. These results suggest that interaction of the Upf1p with the peptidyl release factors may be a key event in the assembly of the putative surveillance complex that enhances translation termination, monitors whether termination has occurred prematurely, and promotes degradation of aberrant transcripts.

ATP is a cofactor of the Upf1 protein that modulates its translation termination and RNA binding activities.

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, ATPgammaS, 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.

Peptidyl-transferase inhibitors have antiviral properties by altering programmed -1 ribosomal frameshifting efficiencies: development of model systems.

The effects of two peptidyl-transferase inhibitors, anisomycin and sparsomycin, on ribosomal frameshifting efficiencies and the propagation of yeast double-stranded RNA viruses were examined. At sublethal doses in yeast cells these drugs specifically alter the efficiency of -1, but not of +1, ribosomal frameshifting. These compounds promote loss of the yeast L-A double-stranded RNA virus, which uses a programmed -1 ribosomal frameshift to produce its Gag-Pol fusion protein. Both of these drugs also change the efficiency of -1 ribosomal frameshifting in yeast and mammalian in vitro translation systems, suggesting that they may have applications to control the propagation of viruses of higher eukaryotes, which also use this translational regulatory mechanism. Our results offer a new set of antiviral agents that may potentially have a broad range of applications in the clinical, veterinary, and agricultural fields.

Making sense of nonsense in yeast.

Messenger RNA (mRNA) degradation is a process that plays an important role in the regulation of gene expression and can be linked to translation. Study of the nonsense-mediated mRNA decay pathway has greatly aided our understanding of the link between these processes. Evidence indicates that this pathway regulates the abundance of both aberrant and wild-type transcripts. Factors involved in this pathway have been identified and recent results indicate that they might also be involved in modulating translation. Here, we discuss the mechanism of nonsense-mediated mRNA decay in the yeast Saccharomyces cerevisiae and the potential role that this pathway can have on the regulation of gene expression.

Identification and characterization of mutations in the UPF1 gene that affect nonsense suppression and the formation of the Upf protein complex but not mRNA turnover.

To understand the relationship between translation and mRNA decay, we have been studying how premature translation termination accelerates the degradation of mRNAs. In the yeast Saccharomyces cerevisiae, the Upf1 protein (Upf1p), which contains a cysteine- and histidine-rich region and nucleoside triphosphate hydrolysis and helicase motifs, was shown to be a trans-acting factor in this decay pathway. A UPF1 gene disruption results in the stabilization of nonsense-containing mRNAs and leads to a nonsense suppression phenotype. Biochemical analysis of the wild-type Upf1p demonstrated that it has RNA-dependent ATPase, RNA helicase, and RNA binding activities. In the work described in the accompanying paper (Y. Weng, K. Czaplinski, and S. W. Peltz, Mol. Cell. Biol. 16:5477-5490, 1996) mutations in the helicase region of Upf1p that inactivated its mRNA decay function but prevented suppression of leu2-2 and tyr7-1 nonsense alleles are identified. On the basis of these results, we suggested that Upf1p is a multifunctional protein involved in modulating mRNA decay and translation termination at nonsense codons. If this is true, we predict that UPF1 mutations with the converse phenotype should be identified. In this report, we describe the identification and biochemical characterization of mutations in the amino-terminal cysteine- and histidine-rich region of Upf1p that have normal nonsense-mediated mRNA decay activities but are able to suppress leu2-2 and tyr7-1 nonsense alleles. Biochemical characterization of these mutant proteins demonstrated that they have altered RNA binding properties. Furthermore, using the two-hybrid system, we characterized the Upf1p-Upf2p interactions and demonstrated that Upf2p interacts with Upf3p. Mutations in the cysteine- and histidine-rich region of Upf1p abolish Upf1p-Upf2p interaction. On the basis of these results, the role of the Upf complex in nonsense-mediated mRNA decay and nonsense suppression is discussed.

Genetic and biochemical characterization of mutations in the ATPase and helicase regions of the Upf1 protein.

mRNA degradation is an important control point in the regulation of gene expression and has been linked to the process of translation. One clear example of this linkage is the nonsense-mediated mRNA decay pathway, in which nonsense mutations in a gene can reduce the abundance of the mRNA transcribed from that gene. For the yeast Saccharomyces cerevisiae, the Upf1 protein (Upf1p), which contains a cysteine- and histidine-rich region and nucleoside triphosphate hydrolysis and helicase motifs, was shown to be a trans-acting factor in this decay pathway. Biochemical analysis of the wild-type Upf1p demonstrates that it has RNA-dependent ATPase, RNA helicase, and RNA binding activities. A UPF1 gene disruption results in stabilization of nonsense-containing mRNAs, leading to the production of enough functional product to overcome an auxotrophy resulting from a nonsense mutation. A genetic and biochemical study of the UPF1 gene was undertaken in order to understand the mechanism of Upf1p function in the nonsense-mediated mRNA decay pathway. Our analysis suggests that Upf1p is a multifunctional protein with separable activities that can affect mRNA turnover and nonsense suppression. Mutations in the conserved helicase motifs of Upf1p that inactivate its mRNA decay function while not allowing suppression of leu2-2 and tyr7-1 nonsense alleles have been identified. In particular, one mutation located in the ATP binding and hydrolysis motif of Upf1p that changed the aspartic and glutamic acid residues to alanine residues (DE572AA) lacked ATPase and helicase activities, and the mutant formed a Upf1p:RNA complex in the absence of ATP; surprisingly, however, the Upf1p:RNA complex dissociated as a consequence of ATP binding. This result suggests that ATP binding, independent of its hydrolysis, can modulate Upf1p:RNA complex formation for this mutant protein. The role of the RNA binding activity of Upf1p in modulating nonsense suppression is discussed.

Purification and characterization of the Upf1 protein: a factor involved in translation and mRNA degradation.

mRNA degradation is an important control point in the regulation of gene expression and has been shown to be linked to the process of translation. One clear example of this linkage is the observation that nonsense mutations in a gene can accelerate the decay of the corresponding mRNA. In the yeast Saccharomyces cerevisiae, the product of the UPF1 gene, harboring zinc finger, NTP hydrolysis, and helicase motifs, was shown to be a trans-acting factor in this decay pathway. A UPF1 gene disruption results in stabilization of nonsense-containing mRNAs and leads to a nonsense suppression phenotype. As a first step toward understanding the molecular and biochemical mechanism of nonsense-mediated mRNA decay, we have purified Upf1p from a yeast extract and characterized its nucleic acid-dependent NTPase activity, helicase activity, and nucleic acid binding properties. The results presented in this paper demonstrate that Upf1p contains both RNA- and DNA-dependent ATPase activities and RNA and DNA helicase activities. In the absence of ATP, Upf1p binds to single-stranded RNA or DNA, whereas hydrolysis of ATP facilitates its release from single-stranded nucleic acid. Based on these results, the role of Upf1p's biochemical activities in mRNA decay and translation are discussed.

Indications of early obesity in low-income Hmong children.

OBJECTIVE: To determine if there is evidence of obesity in low-income Hmong children. DESIGN: Cross-sectional survey. SETTING: Women, Infants, and Children clinics in Minneapolis, Minn. PARTICIPANTS: 271 US-born Hmong children, ages 1.00 through 4.99 years. SELECTION PROCEDURES: Consecutive sample of all Hmong children seen in two clinics between September and December 1989. INTERVENTIONS: None. MEASUREMENTS/MAIN RESULTS: Heights and weights were measured and converted to National Center for Health Statistics z scores of weight for age, height for age, and weight for height. Relative to National Center for Health Statistics reference data, mean height-for-age z scores decline progressively after age 2 years to--1.2 z at age 4.5 years. Mean weight-for-height z scores exceeded the National Center for Health Statistics reference significantly at ages 3 and 4 years, and there was more than a fourfold excess of Hmong children beyond the 95th percentile in weight for height at these ages. CONCLUSIONS: There is evidence of early obesity in Hmong children, an ethnic group heretofore considered to be a low risk for obesity.