The invention relates to nonsense-mediated mRNA decay function.
It is well known in the field of biology that changes in the amino acid sequence of a protein can result in changes in the biological function of the protein. To optimize a target biological function, the amino acid sequence can be altered and tested for improved function. In very simple terms, this is the process of evolution by which the proteins that exist naturally today have been selected over eons. It is an advantage of modern molecular biology that such alterations can be made in a matter of days rather than a matter of centuries. Specifically, optimizing the biological function of a protein of pharmaceutical or other commercial interest can be performed by substituting one amino acid for the naturally occurring amino acid at a given site and producing a sufficient quantity of the protein for screening of biological activity.
Production of a recombinant protein in a cellular system requires the efficient translation of the mRNA transcript encoding the protein. For this to occur, the transcript must exist in the cell long enough for translation into the desired recombinant protein. mRNA transcripts vary in the length of time (transcript half-life) that they exist in a cell prior to being degraded by cellular proteins specific for that purpose. In some cases, degradation occurs rapidly such that very little protein is produced.
For example, the yeast cell, Saccharomyces cerevisiae, a commonly used cellular system for the production of recombinant proteins, has a biological pathway that specifically degrades mRNA transcripts containing a non-coding triplet sequence (nonsense or stop codons) in the transcript. In several genes studied thus far, the destabilizing nonsense codon occurs within the 5'-proximal portion of the transcript (reviewed in Peltz et al., Prog. Nucl. Acids Res. Mol. Biol. (1994) 47:271-297). The translation process stops at the nonsense codons prior to reaching the end of the transcript's coding sequence resulting in the production of a truncated protein that may not possess normal biological activity. Thus, the cell has developed a biochemical system to degrade transcripts containing mutations that create stop codons early in the coding sequence.
However, in a cell of a suppressor strain that suppresses nonsense codons, a nonsense codon can be a useful means of coding for an alternate amino acid when a nonsense codon is engineered into the coding sequence to produce an altered protein which is then screened for enhanced biological activity. Suppressor strains (e.g., SUF1-1) do not allow maximal expression of a nonsense codon-containing transcript (Leeds et al., (1991) Genes & Dev. 5:2303-2314).
Nonsense-mediated mRNA decay is a phenomenon in which nonsense mutations, e.g., point or frame shift mutations that create a stop codon in the reading frame, in a gene can enhance the decay rate of the mRNA transcribed from that gene. For a review, see, e.g., Peltz et al., (1994) Prog. Nuc. Acid Res. Mol. Biol. 47:271-297. The process occurs in viruses, prokaryotes, and eukaryotes (Leeds (1991), supra; Barker, G. F. and Beemon, K. (1991) Mol. Cell. Biol. 11:2760-2768; Lim, S.-K. and Maquat, L. E. (1992) EMBO J. 11:3271-3278).
In most genetic systems, 61 of the 64 possible codon triplets encode amino acids. The triplets UAA, UAG, and UGA are non-coding (nonsense codons) and promote translational termination (Osawa et al., (1992) Microbiol. Rev. 56:229-264). The polypeptide chain terminating effects of UAA, UAG, and UGA triplets have been amply documented and characterized (Craigen et al., (1990) Mol. Microbiol. 4:861-865).
Nonsense-mediated mRNA decay has been studied extensively in the yeast Saccharomyces cerevisiae where it has been shown that degradation of mRNA via this pathway is most likely to occur in the cytoplasm and is linked to translation. Evidence in support of these conclusions includes the following: 1) unstable, nonsense-containing mRNAs are stabilized in a strain harboring an amber suppressor tRNA (Losson and Lacroute, (1979) Proc. Nat'l. Acad. Sci. USA 76:5134-5137; Gozalbo and Hohmann, (1990) Curr. Genet. 17:77-79); 2) nonsense-containing mRNAs are ribosome-associated (Leeds et al., (1991) Genes & Dev. 5:2303-2314; He et al., (1993) Proc. Nat'l. Acad. Sci. USA 90:7034-7039) and the number of ribosomes associated with such mRNAs is a function of the relative positions of the respective nonsense codons (He et al., (1993) Proc. Nat'l. Acad. Sci. USA 90:7034-7039); and 3) treatment of cells with cycloheximide, an inhibitor of translational elongation, stabilizes nonsense-containing mRNAs, yet removal of cycloheximide leads to the immediate restoration of rapid mRNA decay (Peltz et al., (1997) RNA 3:234-244).
Previous studies of nonsense-mediated mRNA decay in yeast also have shown that the products of the UPF1 and UPF3 genes (proteins Upf1p and Upf3p, respectively) are essential components of this degradative pathway. Mutations in these genes stabilize mRNAs containing premature nonsense codons without affecting the decay rates of most wild-type transcripts (Leeds et al., (1991) Genes & Dev. 5:2303-2314, Leeds et al., (1992) Mol. Cell. Biol. 12:2165-2177; Peltz et al., (1993) Genes & Dev. 7:1737-1754; He et al., (1993) Proc. Nat'l. Acad. Sci. USA 90:7034:7039; Cui et al., (1995) Genes & Dev. 9:423-436; He and Jacobson, (1995) Genes & Dev. 9:437-454; He et al., (1997) Mol. Cell. Biol. 17:1580-1594; Lee and Culbertson, (1995) Proc. Nat'l. Acad. Sci. USA 92:10354-10358; Lee and Varmus, (1995) Proc. Nat'l. Acad. Sci. USA 92:6587-6591).
The UPF1 gene has been cloned and sequenced, (Leeds et al., (1992) Mol. Cell Biol. 12:2165-2177) and shown to be: 1) non-essential for viability; 2) capable of encoding a 109 kD protein with a so-called zinc finger, nucleotide (GTP) binding site, and RNA helicase motifs (Leeds et al., (1992) Mol. Cell. Biol. 12:2165-2177; Altamura et al., (1992) J. Mol. Biol. 224:575-587; Koonin, (1992) Trends Biochem. Sci. 17:495-497); 3) identical to NAM7, a nuclear gene that was isolated as a high copy suppressor of mitochondrial RNA splicing mutations (Altamura et al., (1992) J. Mol. Biol. 224:575-587); and 4) partially homologous to the yeast SEN1 gene (Leeds et al., (1992) Mol. Cell. Biol. 12:2165-2177). The latter encodes a noncatalytic subunit of the tRNA splicing endonuclease complex (Winey and Culbertson, (1988) Genetics 118:607-617; DeMarin et al., (1992) Mol. Cell. Biol. 12:2154-2164), suggesting that the Upf1p protein (Upf1p) may also be part of a nuclease complex targeted specifically to nonsense-containing mRNAs.
Suppression of nonsense-mediated mRNA decay in upf1 deletion strains does not appear to result simply from enhanced read-through of the termination signal (Leeds et al., (1991) Genes & Dev. 5:2303-2314), nor does it appear to be specific for a single nonsense codon. The ability of upf1.sup.- mutants to suppress tyr7-1 (UAG), leu2-1 (UAA), leu2-2 (UGA), met8-1 (UAG), and his4-166 (UGA) (Leeds et al., (1992) Mol. Cell. Biol. 12:2165-2177) indicates that they can act as omnipotent suppressors. upf1.sup.- mutants degrade nonsense-containing transcripts at a slower rate allowing synthesis of sufficient read-through protein to permit cells to grow under nutrient-deficient conditions that are nonpermissive for UPF1.sup.+ cells.