A mutated human homologue to yeast Upf1 protein has a dominant-negative effect on the decay of nonsense-containing 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 nonsense-containing 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 nonsense-mediated 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 dominant-negative 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 nonsense-mediated mRNA decay is related mechanistically in yeast and in mammalian cells, regardless of the cellular site of decay.

Figure 1

Transient expression of R(844)C hUPF1 cDNA in COS cells abrogates the nonsense-mediated decrease in the abundance of β-globin mRNA. COS cells were transiently transfected with a pmCMV-Gl test plasmid (either Norm, which is nonsense-free, or 39Ter, which harbors a nonsense codon at amino acid position 39), the phCMV-MUP reference plasmid, and pCI-neo-hUPF1 [either Wt, which harbors an unmutagenized hUpf1p reading frame, or R(844)C, which harbors the arginine-to-cysteine change at amino acid position 844]. The amounts of each plasmid used were, respectively, 10 μg, 3 μg, and 37 μg, or 19 μg, 3 μg, and 28 μg. An appropriate amount of a fourth plasmid was added to each transfection to bring the total amount of introduced DNA to 50 μg. Nuclear and cytoplasmic RNA was purified (29, 30), and 40 μg were analyzed by blot hybridization to detect globin (Gl) RNA and mouse major urinary protein (MUP) RNA. Hybridization was quantitated by PhosphorImaging. The level of Gl mRNA from each mCMV-Gl allele was normalized to the level of MUP mRNA to provide a quantitative analysis. Normalized values then were calculated as a percentage of the normalized value of Gl Norm mRNA in the presence of either the Wt hUPF1 gene or the R844C hUPF1 gene or as a percentage of the normalized value of Gl 39Ter mRNA in the presence of the R844C hUPF1 gene, each of which was considered as 100. Percentages differed between two independently performed experiment by no more than 7%.

Figure 2

Analysis of the amounts of hUPF1 RNA and hUpf1 protein in COS cells. (A) Cytoplasmic RNA (2.5 μg) from the same transfections analyzed in Fig. 1 (lanes 1–4) and Fig. 3 (lanes 12–17) was used to quantitate the levels of hUPF1 RNA and COS cell UPF1 RNA (A) and hUPF1 and MUP RNAs (B) by using RT-PCR. (A) The ratio of RT-PCR products from hUPF1 and COS cell UPF1 RNAs for those transfections involving pCI-neo-hUPF1 R844C was calculated after digestion with HaeIII, which does not cleave the 193-bp RT-PCR product of R844C hUPF1 RNA but generates fragments of 138 bp and 55 bp from the 193-bp RT-PCR product of COS-cell UPF1 RNA. HaeIII cleavage of the RT-PCR product of cells transfected with pCI-neo-hUPF1 Wt (lane 1), like HaeIII cleavage of the RT-PCR product of COS cell UPF1 RNA (lane 5), generates 138-bp and 55-bp fragments. The lanes marked with a wedge constitute the analysis of a serial dilution of cytoplasmic RNA and establish that there is a linear relationship between the amount of input RNA and the amount of each RT-PCR product. Indication that HaeIII cleavage was complete derives from a comparison of lanes 5 and 6 and lanes 16 and 17, i.e., all of the 193-bp product that derives from either COS cell UPF1 RNA or Wt hUPF1 RNA was cleaved by HaeIII to 138-bp and 55-bp fragments. (B) Immunoblot analysis of 30 μg of untransfected cell protein and serial dilutions of 3 μg of transfected COS cell protein using affinity-purified rabbit polyclonal anti-hUpf1p, a secondary peroxidase-conjugated anti-rabbit IgG, and ECL. Dilutions were analyzed to establish that there is a linear relationship between the amounts of input protein and immunoreactive Upf1 protein. Total Upf1p is the sum of hUpf1p and COS-cell Upf1p in 30 μg of total-cell protein, where the level of COS cell Upf1p was considered as 1. Notably, hUpf1p and COS cell Upf1p comigrate.

Figure 3

Transient expression of R844C hUPF1 cDNA in COS cells abrogates the nonsense-mediated decrease in the abundance of GPx1 mRNA. COS cells were transiently transfected with a test plasmid pmCMV-GPx (harboring either a TGC cysteine codon or a TAA nonsense codon at position 46), the reference plasmid phCMV-MUP, and pCI-neo-hUPF1 (harboring either Wt or R844C hUPF cDNA). The amounts of each plasmid used were, respectively, 10 μg, 3 μg, and 37 μg or 10 μg, 3 μg, and 25 μg. An appropriate amount of a fourth plasmid was added to each transfection to bring the total amount of introduced DNA to 50 μg. Nuclear and cytoplasmic RNA was purified, and the amounts of GPx1 and MUP mRNAs were quantitated by using RT-PCR. GPx1 and MUP mRNAs generate 420-bp and 199-bp products, respectively. The level of mRNA from each mCMV-GPx1 allele was normalized to the level of MUP mRNA. Normalized values for GPx1 mRNA harboring UAA(46) then were calculated as a percentage of the normalized value of GPx1 mRNA harboring UGC(46) in the presence of either the Wt hUPF1 gene or the R844C hUPF1 gene, each of which was considered as 100. Percentages differed between two independently performed experiments by no more than 9%.

Figure 4

Stable expression of hUPF1 R(844)C cDNA in HeLa cells abrogates the nonsense-mediated decrease in the abundance of β-globin mRNA. (A) A HeLa cell line stably transfected with pFlag-hUPF1-IRES1neo R844C was transiently transfected with 25 μg of pmCMV-Gl test plasmid (either Norm or 39Ter) and 18 μg of the phCMV-MUP reference plasmid. Total-cell RNA was purified (29, 30), and RT-PCR and PhosphorImaging were used to quantitate Gl and MUP transcripts. Gl and MUP mRNAs generate 486-bp and 199-bp products, respectively. The level of mRNA from each mCMV-Gl allele was normalized to the level of MUP mRNA. Normalized values for Gl mRNA then were calculated as a percentage of the normalized value of Gl Norm mRNA either in the presence (R844C) or absence (−) of the Flag-hUPF1 expression vector, each of which was considered as 100. Percentages differed between two independently performed experiments by no more than 5%. Normalized values for Gl 39Ter mRNA were also calculated as a percentage of the normalized value of Gl 39Ter mRNA in the absence (−) of the Flag-hUPF1 expression vector. (B) Immunoblot analysis of 4.5 μg of protein from untransfected HeLa cells and serial dilutions of 4.5 μg of protein from the HeLa cell line stably transfected with Flag-hUPF1-IRES1neo harboring the R844C mutation. Dilutions were analyzed to establish that there is a linear relationship between the amounts of input protein and immunoreactive hUpf1 protein. Total Upf1p is the sum of R844C hUpf1p and HeLa cell hUpf1p in 4.5 μg of total-cell protein, where the level of hUpf1p was considered as 1.