Frameshift suppressor mutations affecting the major glycine transfer RNAs of Saccharomyces cerevisiae

Mendenhall, Michael J.; Leeds, Peter; Huang, Fen; Mathison, Lorilee; Zwick, Michael G.; Sleiziz, Christine; Culbertson, Michael R.

doi:10.1016/0022-2836(87)90714-5

Cited by 31 publications

(20 citation statements)

References 57 publications

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“…SUF4 glycine tRNA was used as a loading control and was detected with complementary oligonucleotide SUF4A (Table 1). SUF4 tRNA is encoded by an intronless tRNA gene (24). The abundance of SUF4 tRNA is unaffected by loss of SEN1 function.…”

Section: Strains and Media The Strains Used In This Study Were As Fomentioning

confidence: 99%

The Putative Nucleic Acid Helicase Sen1p Is Required for Formation and Stability of Termini and for Maximal Rates of Synthesis and Levels of Accumulation of Small Nucleolar RNAs in Saccharomyces cerevisiae

Rasmussen

Culbertson

1998

Molecular and Cellular Biology

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Sen1p from Saccharomyces cerevisiae is a nucleic acid helicase related to DEAD box RNA helicases and type I DNA helicases. The temperature-sensitive sen1-1 mutation located in the helicase motif alters the accumulation of pre-tRNAs, pre-rRNAs, and some small nuclear RNAs. In this report, we show that cells carrying sen1-1 exhibit altered accumulation of several small nucleolar RNAs (snoRNAs) immediately upon temperature shift. Using Northern blotting, RNase H cleavage, primer extension, and base compositional analysis, we detected three forms of the snoRNA snR13 in wild-type cells: an abundant TMG-capped 124-nucleotide (nt) mature form (snR13F) and two less abundant RNAs, including a heterogeneous population of ϳ1,400-nt 3-extended forms (snR13R) and a 108-nt 5-truncated form (snR13T) that is missing 16 nt at the 5 end. A subpopulation of snR13R contains the same 5 truncation. Newly synthesized snR13R RNA accumulates with time at the expense of snR13F following temperature shift of sen1-1 cells, suggesting a possible precursorproduct relationship. snR13R and snR13T both increase in abundance at the restrictive temperature, indicating that Sen1p stabilizes the 5 end and promotes maturation of the 3 end. snR13F contains canonical C and D boxes common to many snoRNAs. The 5 end of snR13T and the 3 end of snR13F reside within C 2 U 4 sequences that immediately flank the C and D boxes. A mutation in the 5 C 2 U 4 repeat causes underaccumulation of snR13F, whereas mutations in the 3 C 2 U 4 repeat cause the accumulation of two novel RNAs that migrate in the 500-nt range. At the restrictive temperature, double mutants carrying sen1-1 and mutations in the 3 C 2 U 4 repeat show reduced accumulation of the novel RNAs and increased accumulation of snR13R RNA, indicating that Sen1p and the 3 C 2 U 4 sequence act in a common pathway to facilitate 3 end formation. Based on these findings, we propose that Sen1p and the C 2 U 4 repeats that flank the C and D boxes promote maturation of the 3 terminus and stability of the 5 terminus and are required for maximal rates of synthesis and levels of accumulation of mature snR13F.snoRNAs (small nucleolar RNAs) are required for processing and posttranscriptional modification of rRNA (22,23,36). Several snoRNAs facilitate endonucleolytic cleavages in prerRNA. In addition, numerous snoRNAs that contain C and D boxes, short conserved sequences found near snoRNA termini, are involved in the formation of 2Ј-O-methylribose residues in rRNA (7,8,17,35,37). Other snoRNAs that contain the sequence ACA near their 3Ј ends facilitate the formation of pseudouridine in rRNA (25). Many yeast snoRNAs contain trimethylguanosine (TMG) caps at their 5Ј ends (23). TMG caps are hypermethylated forms of monomethylguanosine caps that are added cotranscriptionally to the 5Ј end of RNA polymerase II primary transcripts. The presence of a TMG cap may function to protect the 5Ј ends of snoRNAs from exonucleolytic decay.Most snoRNAs are synthesized via RNA maturation pathways that convert pre-snoRNAs to mature sno...

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Section: Strains and Media The Strains Used In This Study Were As Fomentioning

confidence: 99%

The Putative Nucleic Acid Helicase Sen1p Is Required for Formation and Stability of Termini and for Maximal Rates of Synthesis and Levels of Accumulation of Small Nucleolar RNAs in Saccharomyces cerevisiae

Rasmussen

Culbertson

1998

Molecular and Cellular Biology

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“…Plasmid pMM54 (sufl +, URA3; Mendenhall et al 1987) was linearized by digestion with BglII, which cuts in the sufl +-flanking region, and used to transform strain PLY36 {Table 1). Ura + transformants all contained the chromosomal copy of SUFI-I and were therefore His + at 30~ due to suppression of his4-38.…”

Section: Strain Constructionmentioning

confidence: 99%

“…The mutations arose in a strain containing his4-38, a + 1 frameshift mutation near the 5' end of the HIS4 transcript that causes translational termination at an adjacent downstream stop codon (Donahue et al 1981). In this study we show that the his4-38 mutation results in a four-to fivefold decrease in mRNA stability.In addition to his4-38, the strain used to select mutants also contained SUFI-1, which codes for a glycine tRNA frameshift suppressor that promotes a low level of readthrough of the frameshift mutation by decoding a 4-base codon (Mendenhall et al 1987). Strains carrying both his4-38 and SUFI-1 have a His + phenotype at 30~…”

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The product of the yeast UPF1 gene is required for rapid turnover of mRNAs containing a premature translational termination codon.

Leeds¹,

Peltz²,

Jacobson³

et al. 1991

Genes & Development

446

462

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mRNA decay rates often increase when translation is terminated prematurely due to a frameshift or nonsense mutation. We have identified a yeast gene, UPF1, that codes for a trans-acting factor whose function is necessary for enhanced turnover of mRNAs containing a premature stop codon. In the absence of UPF1 function, frameshift or nonsense mutations in the HIS4 or LEU2 genes that normally cause rapid mRNA decay fail to have this effect. Instead, the mRNAs decay at rates similar to the corresponding wild-type mRNAs. The stabilization of frameshift or nonsense mRNAs observed in upfl-strains does not appear to result from enhanced readthrough of the termination signal. Loss of UPF1 function has no effect on the accumulation or stability of HIS4 + or LEU2 + mRNA, suggesting that the UPF1 product functions only in response to a premature termination signal. When we examined the accumulation and stability of other wild-type mRNAs in the presence or absence of UPF1, including MAT~I, STE3, ACT1, PGK1, PAB1, and URA3 mRNAs, only the URA3 transcript was affected. On the basis of these and other results, the UPF1 product appears to participate in a previously uncharacterized pathway leading to the degradation of a limited class of yeast transcripts. Nonsense mutations that generate a premature translational termination signal often reduce the steady-state accumulation of the corresponding mRNA (Brown 1989;Peltz et al. 1990). In a study of the yeast URA3 gene, it was shown that the extent of reduced mRNA accumulation depends on the position of the nonsense mutation (Losson and Lacroute 1979). Mutations near the 5' end of the transcript were shown to have a greater destabilizing effect than mutations near the 3' end. Furthermore, introduction of an amber tRNA suppressor restabilized ura3 nonsense mRNA, indicating that the turnover rate is determined in part by the relative efficiencies of termination versus readthrough of the stop codon. These studies suggested that the turnover rate of nonsense mRNA is probably related to some aspect of its translation rather than to a potential change in mRNA structure that might result from the presence of a nonsense mutation.Similar studies in higher eukaryotes have proven more difficult to interpret. In some cases, the introduction of a premature stop codon into a gene has been linked to 3Corresponding author. increased cytoplasmic turnover (Maquat et al. 1981;Barker and Beemon 1991). However, other studies suggest that nonsense mutations may cause changes in nuclear processing and/or transport, and these changes, rather than cytoplasmic mRNA degradation, may be primarily responsible for decreased steady-state mRNA levels (Humphries et al. 1984;Takeshita et al. 1984;Urlaub et al. 1989;Cheng et al. 1990).Here, we report the characterization of mutations in the yeast Saccharomyces cerevisiae that specifically stabilize mRNAs containing a premature translational termination signal. The mutations arose in a strain containing his4-38, a + 1 frameshift mutation near the 5' end of the HIS4 tr...

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“…The his4-38 mutation results in a four-to fivefold decrease in mRNA stability (29). The strain used to select mutations also contained SUFJ-J, which codes for a glycine tRNA frameshift suppressor that promotes inefficient read-through of the his4-38 frameshift mutation by decoding a four-base codon (36). The SUFI-l tRNA suppressor does not restabilize his4-38 mRNA to a significant extent, probably because of a low efficiency of suppression (29).…”

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confidence: 99%

Gene products that promote mRNA turnover in Saccharomyces cerevisiae.

Leeds¹,

Wood²,

Lee³

et al. 1992

Mol. Cell. Biol.

298

323

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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)...

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Frameshift suppressor mutations affecting the major glycine transfer RNAs of Saccharomyces cerevisiae

Cited by 31 publications

References 57 publications

The Putative Nucleic Acid Helicase Sen1p Is Required for Formation and Stability of Termini and for Maximal Rates of Synthesis and Levels of Accumulation of Small Nucleolar RNAs in Saccharomyces cerevisiae

The Putative Nucleic Acid Helicase Sen1p Is Required for Formation and Stability of Termini and for Maximal Rates of Synthesis and Levels of Accumulation of Small Nucleolar RNAs in Saccharomyces cerevisiae

The product of the yeast UPF1 gene is required for rapid turnover of mRNAs containing a premature translational termination codon.

Gene products that promote mRNA turnover in Saccharomyces cerevisiae.

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