Gene products that promote mRNA turnover in Saccharomyces cerevisiae.

Abstract

We showed previously that the increased rate of mRNA turnover associated with premature translational termination in the yeast Saccharomyces cerevisiae requires a functional UPF1 gene product. In this study, we show that the UPFI gene codes for a 109-kDa primary translation product whose function is not essential for growth. The protein contains a potential zinc-dependent nucleic acid-binding domain and a nucleoside triphosphate-binding domain. A 300-amino-acid segment of the UPF1 protein is 36% identical to a segment of the yeast SEN1 protein, which is required for endonucleolytic processing of intron-containing pre-tRNAs. The same region is 32% identical to a segment of Mov-10, a mouse protein of unknown function. Dominant-negative upfl mutations were isolated following in vitro mutagenesis of a plasmid containing the UPF1 gene. They mapped exclusively at conserved positions within the sequence element common to all three proteins, whereas the recessive upfl-2 mutation maps outside this region. The clustering of dominant-negative mutations suggests the presence of a functional domain in UPF1 that may be shared by all three proteins. We also identified upf mutations in three other genes designated UPF2, UPF3, and UPF4. When alleles of each gene were screened for effects on mRNA accumulation, we found that the recessive mutation upJ3-1 causes increased accumulation of mRNA containing a premature stop codon. When mRNA half-lives were measured, we found that excess mRNA accumulation was due to mRNA stabilization. On the basis of these results, we suggest that the products of at least two genes, UPF1 and UPF3, are responsible for the accelerated rate of mRNA decay associated with premature translational termination.In a wide variety of organisms, mRNAs transcribed from genes containing nonsense or frameshift mutations accumulate to a much lesser extent than do the corresponding wild-type mRNAs. In Saccharomyces cerevisiae, the introduction of a premature stop codon into a transcript causes a reduction in mRNA half-life that leads to a decrease in steady-state mRNA accumulation (29,34,48). The introduction of an efficient tRNA nonsense suppressor, which promotes read-through and restores translation of the mRNA, prevents the decline in stability and accumulation caused by premature translational termination (34). These results suggest the existence of a mechanism that serves to adjust the intrinsic rate of mRNA decay according to the ability of the mRNA to be translated. The underlying molecular basis for such a mechanism has not yet been established.To further study how mRNA turnover is related to premature translational termination, we took advantage of a selection scheme capable of yielding mutations that uncouple the two processes. The mutations were obtained in a strain containing his4-38, a + 1 frameshift near the 5' end of the HIS4 transcript that causes translational termination at an adjacent downstream stop codon (7, 13). The his4-38 mutation results in a four-to fivefold decrease in mRNA stability (29)...

mRNA destabilization triggered by premature translational termination depends on at least three cis-acting sequence elements and one trans-acting factor.

“…The UPF1 gene has been cloned and sequenced and shown to be (1) nonessential for viability, (2) capable of encoding a 109-kD protein with both zinc finger, nucleotide (GTP)-binding site and RNA helicase motifs, (3) identical to NAM7, a nuclear gene that was isolated as a high-copy suppressor of mitochondrial RNA splicing mutations, and (4) partially homologous to the yeast SEN1 gene (Leeds et al 1991(Leeds et al ,1992Altamura et al 1992;Koonin 1992). The latter encodes a noncatalytic subunit of the tRNA splicing endonuclease complex (Winey and Culbertson 1988), suggesting that Upflp may also be part of a nuclease complex targeted specifically to nonsensecontaining mRNAs.…”

“…Previous studies of nonsense-mediated mRNA decay in yeast showed that (1) mRNA destabilization is linked to premature translational termination, because nonsense-containing URA3 mRNA is stabilized in a strain containing an amber suppressor tRNA (Losson and Lacroute 1979); (2) the extent of destabilization is position dependent, because 5' proximal nonsense mutations destabilize transcripts to a greater degree than those that are 3' proximal (Losson and Lacroute 1979;Pelsy and Lacroute 1984;Leeds et al 1991;; and (3) the products of the UPF1 and UPF3 genes are involved in this degradative pathway, as mutations in these genes stabilize mRNAs with nonsense mutations without affecting the half-lives of most wild-type transcripts (Leeds et al 1991(Leeds et al ,1992.…”

Identification and Characterization of a Sequence Motif Involved in Nonsense-Mediated mRNA Decay

“…Reduced mRNA levels or decreased stabilities of nonsense-containing transcripts have been observed in both prokaryotes and eukaryotes (6-8, 13, 14, 18, 20, 22, 42, 44-47, 51, 54, 56, 71, 75). For example, in the yeast S. cerevisiae, a nonsense-containing URA3 mRNA is stabilized in a strain containing an amber suppressor tRNA, indicating that mRNA destabilization is linked to premature translational termination (45).Trans-acting factors involved in nonsense-mediated mRNA decay have been identified in studies undertaken to investigate a class of yeast non-tRNA nonsense suppressors (17,42,43). Mutations in the UPF genes were isolated as allosuppressors of a his4 frameshift allele (17,43).…”

“…Mutations in the UPF genes were isolated as allosuppressors of a his4 frameshift allele (17,43). Subsequent studies demonstrated that strains harboring the upf1 and upf3 alleles, and as shown in more recent studies also upf2, lead to the selective stabilization of nonsense-containing mRNAs without affecting the decay rates of most other mRNAs (16,26,42,43,56).…”

mRNA surveillance in mammalian cells: The relationship between introns and translation termination

“…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, 1995Maquat, , 1996Li & 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+, 1997Moriarty et al+, , 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)+ Based on studies of disease-associated and in vitromutagenized genes, a rule for termination-codon position within intron-containing genes has been established for mammalian cells: only those termination codons located more than 50-55 nt upstream of the 39-most exon-exon junction mediate mRNA decay (Cheng et al+, 1994;Thermann et al+, 1998;Zhang et al+, 1998aZhang et al+, , 1998b)+ In support of this rule, more than 98% of genes having one or more 39-untranslated exons terminate translation less than 50 nt upstream of the 39-most exon-exon junction (Nagy & Maquat, 1998)+ There is a growing consensus that exon-exon junctions function in NMD+ According to current thinking, the process of pre-mRNA splicing influences product mRNP structure and, in so doing, NMD, by leaving a mark at the junctions ...…”