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Mol Cell Biol. 1996 October; 16(10): 5477–5490.
PMCID: PMC231548

Genetic and biochemical characterization of mutations in the ATPase and helicase regions of the Upf1 protein.


mRNA degradation is an important control point in the regulation of gene expression and has been linked to the process of translation. One clear example of this linkage is the nonsense-mediated mRNA decay pathway, in which nonsense mutations in a gene can reduce the abundance of the mRNA transcribed from that gene. For the yeast Saccharomyces cerevisiae, the Upf1 protein (Upf1p), which contains a cysteine- and histidine-rich region and nucleoside triphosphate hydrolysis and helicase motifs, was shown to be a trans-acting factor in this decay pathway. Biochemical analysis of the wild-type Upf1p demonstrates that it has RNA-dependent ATPase, RNA helicase, and RNA binding activities. A UPF1 gene disruption results in stabilization of nonsense-containing mRNAs, leading to the production of enough functional product to overcome an auxotrophy resulting from a nonsense mutation. A genetic and biochemical study of the UPF1 gene was undertaken in order to understand the mechanism of Upf1p function in the nonsense-mediated mRNA decay pathway. Our analysis suggests that Upf1p is a multifunctional protein with separable activities that can affect mRNA turnover and nonsense suppression. Mutations in the conserved helicase motifs of Upf1p that inactivate its mRNA decay function while not allowing suppression of leu2-2 and tyr7-1 nonsense alleles have been identified. In particular, one mutation located in the ATP binding and hydrolysis motif of Upf1p that changed the aspartic and glutamic acid residues to alanine residues (DE572AA) lacked ATPase and helicase activities, and the mutant formed a Upf1p:RNA complex in the absence of ATP; surprisingly, however, the Upf1p:RNA complex dissociated as a consequence of ATP binding. This result suggests that ATP binding, independent of its hydrolysis, can modulate Upf1p:RNA complex formation for this mutant protein. The role of the RNA binding activity of Upf1p in modulating nonsense suppression is discussed.

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Selected References

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  • Aharon T, Schneider RJ. Selective destabilization of short-lived mRNAs with the granulocyte-macrophage colony-stimulating factor AU-rich 3' noncoding region is mediated by a cotranslational mechanism. Mol Cell Biol. 1993 Mar;13(3):1971–1980. [PMC free article] [PubMed]
  • Altamura N, Groudinsky O, Dujardin G, Slonimski PP. NAM7 nuclear gene encodes a novel member of a family of helicases with a Zn-ligand motif and is involved in mitochondrial functions in Saccharomyces cerevisiae. J Mol Biol. 1992 Apr 5;224(3):575–587. [PubMed]
  • Atkin AL, Altamura N, Leeds P, Culbertson MR. The majority of yeast UPF1 co-localizes with polyribosomes in the cytoplasm. Mol Biol Cell. 1995 May;6(5):611–625. [PMC free article] [PubMed]
  • Brosh RM, Jr, Matson SW. Mutations in motif II of Escherichia coli DNA helicase II render the enzyme nonfunctional in both mismatch repair and excision repair with differential effects on the unwinding reaction. J Bacteriol. 1995 Oct;177(19):5612–5621. [PMC free article] [PubMed]
  • Caponigro G, Muhlrad D, Parker R. A small segment of the MAT alpha 1 transcript promotes mRNA decay in Saccharomyces cerevisiae: a stimulatory role for rare codons. Mol Cell Biol. 1993 Sep;13(9):5141–5148. [PMC free article] [PubMed]
  • Cleveland DW. Autoregulated instability of tubulin mRNAs: a novel eukaryotic regulatory mechanism. Trends Biochem Sci. 1988 Sep;13(9):339–343. [PubMed]
  • Cui Y, Hagan KW, Zhang S, Peltz SW. Identification and characterization of genes that are required for the accelerated degradation of mRNAs containing a premature translational termination codon. Genes Dev. 1995 Feb 15;9(4):423–436. [PubMed]
  • Culbertson MR, Underbrink KM, Fink GR. Frameshift suppression Saccharomyces cerevisiae. II. Genetic properties of group II suppressors. Genetics. 1980 Aug;95(4):833–853. [PubMed]
  • Curatola AM, Nadal MS, Schneider RJ. Rapid degradation of AU-rich element (ARE) mRNAs is activated by ribosome transit and blocked by secondary structure at any position 5' to the ARE. Mol Cell Biol. 1995 Nov;15(11):6331–6340. [PMC free article] [PubMed]
  • Czaplinski K, Weng Y, Hagan KW, Peltz SW. Purification and characterization of the Upf1 protein: a factor involved in translation and mRNA degradation. RNA. 1995 Aug;1(6):610–623. [PubMed]
  • Decker CJ, Parker R. A turnover pathway for both stable and unstable mRNAs in yeast: evidence for a requirement for deadenylation. Genes Dev. 1993 Aug;7(8):1632–1643. [PubMed]
  • Decker CJ, Parker R. Mechanisms of mRNA degradation in eukaryotes. Trends Biochem Sci. 1994 Aug;19(8):336–340. [PubMed]
  • de Vos AM, Tong L, Milburn MV, Matias PM, Jancarik J, Noguchi S, Nishimura S, Miura K, Ohtsuka E, Kim SH. Three-dimensional structure of an oncogene protein: catalytic domain of human c-H-ras p21. Science. 1988 Feb 19;239(4842):888–893. [PubMed]
  • Feinberg AP, Vogelstein B. "A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity". Addendum. Anal Biochem. 1984 Feb;137(1):266–267. [PubMed]
  • Ferguson J, Groppe JC, Reed SI. Construction and characterization of three yeast-Escherichia coli shuttle vectors designed for rapid subcloning of yeast genes on small DNA fragments. Gene. 1981 Dec;16(1-3):191–197. [PubMed]
  • Fields S, Song O. A novel genetic system to detect protein-protein interactions. Nature. 1989 Jul 20;340(6230):245–246. [PubMed]
  • Fry DC, Kuby SA, Mildvan AS. ATP-binding site of adenylate kinase: mechanistic implications of its homology with ras-encoded p21, F1-ATPase, and other nucleotide-binding proteins. Proc Natl Acad Sci U S A. 1986 Feb;83(4):907–911. [PubMed]
  • Gay DA, Yen TJ, Lau JT, Cleveland DW. Sequences that confer beta-tubulin autoregulation through modulated mRNA stability reside within exon 1 of a beta-tubulin mRNA. Cell. 1987 Aug 28;50(5):671–679. [PubMed]
  • Graves RA, Pandey NB, Chodchoy N, Marzluff WF. Translation is required for regulation of histone mRNA degradation. Cell. 1987 Feb 27;48(4):615–626. [PubMed]
  • Hagan KW, Ruiz-Echevarria MJ, Quan Y, Peltz SW. Characterization of cis-acting sequences and decay intermediates involved in nonsense-mediated mRNA turnover. Mol Cell Biol. 1995 Feb;15(2):809–823. [PMC free article] [PubMed]
  • He F, Brown AH, Jacobson A. Interaction between Nmd2p and Upf1p is required for activity but not for dominant-negative inhibition of the nonsense-mediated mRNA decay pathway in yeast. RNA. 1996 Feb;2(2):153–170. [PubMed]
  • He F, Jacobson A. Identification of a novel component of the nonsense-mediated mRNA decay pathway by use of an interacting protein screen. Genes Dev. 1995 Feb 15;9(4):437–454. [PubMed]
  • He F, Peltz SW, Donahue JL, Rosbash M, Jacobson A. Stabilization and ribosome association of unspliced pre-mRNAs in a yeast upf1- mutant. Proc Natl Acad Sci U S A. 1993 Aug 1;90(15):7034–7038. [PubMed]
  • Hodgkin J, Papp A, Pulak R, Ambros V, Anderson P. A new kind of informational suppression in the nematode Caenorhabditis elegans. Genetics. 1989 Oct;123(2):301–313. [PubMed]
  • Hodgman TC. A new superfamily of replicative proteins. Nature. 1988 May 5;333(6168):22–23. [PubMed]
  • Ito H, Fukuda Y, Murata K, Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. [PMC free article] [PubMed]
  • Jurnak F. The three-dimensional structure of c-H-ras p21: implications for oncogene and G protein studies. Trends Biochem Sci. 1988 Jun;13(6):195–198. [PubMed]
  • Koonin EV. A new group of putative RNA helicases. Trends Biochem Sci. 1992 Dec;17(12):495–497. [PubMed]
  • la Cour TF, Nyborg J, Thirup S, Clark BF. Structural details of the binding of guanosine diphosphate to elongation factor Tu from E. coli as studied by X-ray crystallography. EMBO J. 1985 Sep;4(9):2385–2388. [PubMed]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
  • Laird-Offringa IA. What determines the instability of c-myc proto-oncogene mRNA? Bioessays. 1992 Feb;14(2):119–124. [PubMed]
  • Lee BS, Culbertson MR. Identification of an additional gene required for eukaryotic nonsense mRNA turnover. Proc Natl Acad Sci U S A. 1995 Oct 24;92(22):10354–10358. [PubMed]
  • Lee CG, Hurwitz J. A new RNA helicase isolated from HeLa cells that catalytically translocates in the 3' to 5' direction. J Biol Chem. 1992 Mar 5;267(7):4398–4407. [PubMed]
  • Leeds P, Peltz SW, Jacobson A, Culbertson MR. The product of the yeast UPF1 gene is required for rapid turnover of mRNAs containing a premature translational termination codon. Genes Dev. 1991 Dec;5(12A):2303–2314. [PubMed]
  • Leeds P, Wood JM, Lee BS, Culbertson MR. Gene products that promote mRNA turnover in Saccharomyces cerevisiae. Mol Cell Biol. 1992 May;12(5):2165–2177. [PMC free article] [PubMed]
  • Linder P, Lasko PF, Ashburner M, Leroy P, Nielsen PJ, Nishi K, Schnier J, Slonimski PP. Birth of the D-E-A-D box. Nature. 1989 Jan 12;337(6203):121–122. [PubMed]
  • Maquat LE. When cells stop making sense: effects of nonsense codons on RNA metabolism in vertebrate cells. RNA. 1995 Jul;1(5):453–465. [PubMed]
  • Muhlrad D, Parker R. Premature translational termination triggers mRNA decapping. Nature. 1994 Aug 18;370(6490):578–581. [PubMed]
  • Parker R, Jacobson A. Translation and a 42-nucleotide segment within the coding region of the mRNA encoded by the MAT alpha 1 gene are involved in promoting rapid mRNA decay in yeast. Proc Natl Acad Sci U S A. 1990 Apr;87(7):2780–2784. [PubMed]
  • Pause A, Sonenberg N. Mutational analysis of a DEAD box RNA helicase: the mammalian translation initiation factor eIF-4A. EMBO J. 1992 Jul;11(7):2643–2654. [PubMed]
  • Peltz SW, Brown AH, Jacobson A. mRNA destabilization triggered by premature translational termination depends on at least three cis-acting sequence elements and one trans-acting factor. Genes Dev. 1993 Sep;7(9):1737–1754. [PubMed]
  • Peltz SW, He F, Welch E, Jacobson A. Nonsense-mediated mRNA decay in yeast. Prog Nucleic Acid Res Mol Biol. 1994;47:271–298. [PubMed]
  • Pinto I, Na JG, Sherman F, Hampsey M. cis- and trans-acting suppressors of a translation initiation defect at the cyc1 locus of Saccharomyces cerevisiae. Genetics. 1992 Sep;132(1):97–112. [PubMed]
  • Pulak R, Anderson P. mRNA surveillance by the Caenorhabditis elegans smg genes. Genes Dev. 1993 Oct;7(10):1885–1897. [PubMed]
  • Ross J. mRNA stability in mammalian cells. Microbiol Rev. 1995 Sep;59(3):423–450. [PMC free article] [PubMed]
  • Rozen F, Edery I, Meerovitch K, Dever TE, Merrick WC, Sonenberg N. Bidirectional RNA helicase activity of eucaryotic translation initiation factors 4A and 4F. Mol Cell Biol. 1990 Mar;10(3):1134–1144. [PMC free article] [PubMed]
  • Ruiz-Echevarria MJ, Peltz SW. Utilizing the GCN4 leader region to investigate the role of the sequence determinants in nonsense-mediated mRNA decay. EMBO J. 1996 Jun 3;15(11):2810–2819. [PubMed]
  • Sachs AB. Messenger RNA degradation in eukaryotes. Cell. 1993 Aug 13;74(3):413–421. [PubMed]
  • Schiavi SC, Wellington CL, Shyu AB, Chen CY, Greenberg ME, Belasco JG. Multiple elements in the c-fos protein-coding region facilitate mRNA deadenylation and decay by a mechanism coupled to translation. J Biol Chem. 1994 Feb 4;269(5):3441–3448. [PubMed]
  • Schiestl RH, Gietz RD. High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Curr Genet. 1989 Dec;16(5-6):339–346. [PubMed]
  • Schmid SR, Linder P. D-E-A-D protein family of putative RNA helicases. Mol Microbiol. 1992 Feb;6(3):283–291. [PubMed]
  • Stansfield I, Tuite MF. Polypeptide chain termination in Saccharomyces cerevisiae. Curr Genet. 1994 May;25(5):385–395. [PubMed]
  • Thomas PS. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5201–5205. [PubMed]
  • Venkatesan M, Silver LL, Nossal NG. Bacteriophage T4 gene 41 protein, required for the synthesis of RNA primers, is also a DNA helicase. J Biol Chem. 1982 Oct 25;257(20):12426–12434. [PubMed]
  • Weng Y, Czaplinski K, Peltz SW. Identification and characterization of mutations in the UPF1 gene that affect nonsense suppression and the formation of the Upf protein complex but not mRNA turnover. Mol Cell Biol. 1996 Oct;16(10):5491–5506. [PMC free article] [PubMed]
  • Winstall E, Gamache M, Raymond V. Rapid mRNA degradation mediated by the c-fos 3' AU-rich element and that mediated by the granulocyte-macrophage colony-stimulating factor 3' AU-rich element occur through similar polysome-associated mechanisms. Mol Cell Biol. 1995 Jul;15(7):3796–3804. [PMC free article] [PubMed]
  • Wisdom R, Lee W. The protein-coding region of c-myc mRNA contains a sequence that specifies rapid mRNA turnover and induction by protein synthesis inhibitors. Genes Dev. 1991 Feb;5(2):232–243. [PubMed]
  • Yun DF, Sherman F. Initiation of translation can occur only in a restricted region of the CYC1 mRNA of Saccharomyces cerevisiae. Mol Cell Biol. 1995 Feb;15(2):1021–1033. [PMC free article] [PubMed]
  • Zhang S, Ruiz-Echevarria MJ, Quan Y, Peltz SW. Identification and characterization of a sequence motif involved in nonsense-mediated mRNA decay. Mol Cell Biol. 1995 Apr;15(4):2231–2244. [PMC free article] [PubMed]

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