A mutated human homologue to yeast Upf1 protein has a dominant-negative effect on the decay of nonsensecontaining mRNAs in mammalian cells

Abstract

All eukaryotic cells analyzed have developed mechanisms to eliminate the production of mRNAs that prematurely terminate translation. The mechanisms are thought to exist to protect cells from the deleterious effects of in-frame nonsense codons that are generated by routine inefficiencies and inaccuracies in RNA metabolism such as pre-mRNA splicing. Depending on the particular mRNA and how it is produced, nonsense codons can mediate a reduction in mRNA abundance either (i) before its release from an association with nuclei into the cytoplasm, presumably but not certainly while the mRNA is being exported to the cytoplasm and translated by cytoplasmic ribosomes, or (ii) in the cytoplasm. Here, we provide evidence for a factor that functions to eliminate the production of nonsensecontaining RNAs in mammalian cells. The factor, variously referred to as Rent1 (regulator of nonsense transcripts) or HUPF1 (human Upf1 protein), was identified by isolating cDNA for a human homologue to Saccharomyces cerevisiae Upf1p, which is a group I RNA helicase that functions in the nonsensemediated decay of mRNA in yeast. Using monkey COS cells and human HeLa cells, we demonstrate that expression of human Upf1 protein harboring an arginine-to-cysteine mutation at residue 844 within the RNA helicase domain acts in a dominantnegative fashion to abrogate the decay of nonsense-containing mRNA that takes place (i) in association with nuclei or (ii) in the cytoplasm. These findings provide evidence that nonsensemediated mRNA decay is related mechanistically in yeast and in mammalian cells, regardless of the cellular site of decay.All eukaryotic cells are thought to be characterized by pathways that eliminate the production of mRNAs that encode truncated proteins (reviewed in refs. 1-4). Truncated proteins can arise as the consequence of inefficiencies or inaccuracies in RNA metabolism. In fact, errors in transcription initiation and splicing can result in the production of either nonfunctional protein or protein that acts in a dominant-negative or gain-of-function fashion and may have provided the selective pressure under which the nonsense-mediated decay pathway evolved (reviewed in ref.2). The nonsense-mediated decay pathway also eliminates nonsensecontaining mRNAs for Igs and T cell receptors that arise as a consequence of nonproductive gene rearrangements and hypermutations (reviewed in ref. 5), certain selenoprotein mRNAs when a UGA codon is recognized as a premature termination codon rather than a selenocysteine codon (6), and nonsensecontaining mRNAs that arise as the consequence of random germ-line or somatic defects (reviewed in ref.2).Trans-acting factors required for nonsense-mediated mRNA decay in S. cerevisiae have been defined by genetic analyses to include Upf1p͞Isf2p͞Sal2p͞Nam7p, Upf2p͞Nmd2p͞Sua1p͞Ifs1p, Upf3p͞Sua6p, Xrn1p, which is a 5Ј 3 3Ј exonuclease, and Dcp1p, which is a decapping enzyme (7-19). Evidence that the three Upf factors function in a common pathway that leads to the accelerated decay of nonsense-cont...

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

“…The RNA-binding characteristics of HUpf1 were investigated using a gel shift assay described previously (Czaplinski et al+, 1995;Weng et al+, 1996aWeng et al+, , 1996b)+ A uniformly labeled 130-nt-long transcript generated from random sequence was used as a probe+ The ability of HUpf1 protein to bind this RNA was assayed by monitoring the mobility of the RNA probe in a native gel+ The results demonstrated that addition of HUpf1 protein retarded the mobility of the substrate RNA consistent with formation of HUpf1:RNA complexes (Fig+ 6A, lane 2)+ We next asked whether ATP addition to the reactions reduced the HUpf1:RNA complexes+ Reactions were prepared as described above and ATP was subsequently added to the mixtures+ The results demonstrated that addition of ATP to the reactions markedly reduced formation of the HUpf1:RNA complexes (Fig+ 6A, lane 3)+ Taken together, these results demon- (Sun et al+, 1998)+ These results indicate that human Upf1 is an important factor in regulating mRNA decay and translation, and understanding its biochemical activities is important for elucidating its role in NMD and translation termination+ The studies reported here describe the approach used to purify the human Upf1 protein and characterize its biochemical properties+ An epitope-tagged HUpf1 was expressed in a baculovirus expression system and was purified from extracts of insect cells infected with recombinant baculovirus particles+ Purification was achieved in a single step using immunoaffinity chromatography with a monoclonal antibody against the Flag-epitope tag inserted at the amino terminus of the protein+ The purified HUpf1 demonstrated a nucleic-acid-dependent ATPase activity that could be stimulated by several different RNA or DNA polymers (Fig+ 2)+ The ATPase assays were optimized and the results demonstrated that the ATPase activity of HUpf1 functioned best at pH 4-6, with increases in pH leading to reduced activity (Fig+ 2A)+ The ATPase activity was present over a broad range of salt concentrations (0-300 mM Analogous to the yeast Upf1p, the HUpf1 was also shown to be a helicase+ The HUpf1 was able to unwind a partially duplexed nucleic acid substrate+ The yeast Upf1p was shown to displace duplexes containing a 59 single-stranded extension (Czaplinski et al+, 1995 Understanding the biochemical activities of the yeast Upf1p has provided insight into how the Upf1p may influence translation termination and NMD (Weng et al+, 1996a(Weng et al+, , 1996b(Weng et al+, , 1998Czaplinski et al+, 1998Czaplinski et al+, , 1999)+ These results showed that ATP is an important cofactor that can modulate Upf1p RNA-binding activity (Weng et al+, 1998)+ Furthermore, the ATPase/helicase activity of the Upf1p is important for NMD, but is not required for its role in translation termination+ Considering the strong conservation of sequence and function between the yeast and human Upf1, we suggest that these activities will also be important for HUpf1 to function+ Future experiments will be performed to characterize the interactions of the human translation termination factors with the Upf1 and the consequence of these interactions on its biochemical activity+ Furthermore, additional investigations are required to understand the role of the divergent amino-and carboxyl-terminal ends of the HUpf1+ It is possible that these unique domains may be interaction domains for factors that are unique to mammalian organisms that may...…”

Nonsense-mediated Decay of mRNA for the Selenoprotein Phospholipid Hydroperoxide Glutathione Peroxidase Is Detectable in Cultured Cells but Masked or Inhibited in Rat Tissues

“…Second, Se concentration affected only the level of UGA(72)-containing mRNA. Third, the level of UGA(72)-containing mRNA, like UAA(72)-containing mRNA but not UGU(72)-containing mRNA, was increased by the expression of a dominant-negative form of hUpf1 protein (Figure 4), which is known to abrogate NMD (Sun et al, 1998). We conclude that PHGPx mRNA that derives from a full-length gene contains all of the cis-acting sequences required to support NMD.…”

“…Each nonsense codon resulted in a reduction in mRNA abundance (Figure 3). In keeping with the idea that the reduction in abundance is a reflection of NMD, overexpression of a dominant-negative (R844C) form of human (h) Upf1 protein (Sun et al, 1998) increased the levels of PHGPx mRNA harboring either UGA(72) or UAA(72) approximately twofold but had no effect on the level of PHGPx mRNA harboring UGU(72), whereas overexpression of a wild-type (Wt) form of hUpf1 protein had no effect on the level of any of the three mRNAs (Figure 4).One possible explanation for the failure of the transient transfections to recapitulate the metabolism of PHGPx RNA observed for rat liver and testis could be that the transfections were performed with an incomplete PHGPx gene. To .…”

Nonsense surveillance regulates expression of diverse classes of mammalian transcripts and mutes genomic noise

“…This pathway can modulate phenotypes arising from nonsense or frameshift mutations, but little is known about the physiologic role of NMD in higher eukaryotes. To address this issue, we examined expression profiles in mammalian cells depleted of Rent1 (also called hUpf1), a factor essential for NMD 3,4 . Upregulated transcripts included those with upstream open reading frames in the 5¢ untranslated region, alternative splicing that introduces nonsense codons or frameshifts, introns in the 3¢ untranslated region or selenocysteine codons.…”