Identification of stop codon readthrough genes in Saccharomyces cerevisiae.

We specifically sought genes within the yeast genome controlled by a non-conventional translation mechanism involving the stop codon. For this reason, we designed a computer program using the yeast database genomic regions, and seeking two adjacent open reading frames separated only by a unique stop codon (called SORFs). Among the 58 SORFs identified, eight displayed a stop codon bypass level ranging from 3 to 25%. For each of the eight sequences, we demonstrated the presence of a poly(A) mRNA. Using isogenic [PSI(+)] and [psi(-)] yeast strains, we showed that for two of the sequences the mechanism used is a bona fide readthrough. However, the six remaining sequences were not sensitive to the PSI state, indicating either a translation termination process independent of eRF3 or a new stop codon bypass mechanism. Our results demonstrate that the presence of a stop codon in a large ORF may not always correspond to a sequencing error, or a pseudogene, but can be a recoding signal in a functional gene. This emphasizes that genome annotation should take into account the fact that recoding signals could be more frequently used than previously expected.

[1]  I. Brierley,et al.  Mutational analysis of the RNA pseudoknot component of a coronavirus ribosomal frameshifting signal☆ , 1991, Journal of Molecular Biology.

[2]  M. Ehrenberg,et al.  A Posttermination Ribosomal Complex Is the Guanine Nucleotide Exchange Factor for Peptide Release Factor RF3 , 2001, Cell.

[3]  S. Ao,et al.  A persistent untranslated sequence within bacteriophage T4 DNA topoisomerase gene 60. , 1988, Science.

[4]  C. Kahana,et al.  Polyamines regulate the expression of ornithine decarboxylase antizyme in vitro by inducing ribosomal frame-shifting. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[5]  R. Lewin.  Editing mRNA precursors. , 1983, Science.

[6]  O. Namy,et al.  Nonsense-mediated decay mutants do not affect programmed -1 frameshifting. , 2000, RNA.

[7]  J. van Helden,et al.  Statistical analysis of yeast genomic downstream sequences reveals putative polyadenylation signals. , 2000, Nucleic acids research.

[8]  Philip J. Farabaugh,et al.  25 Programmed Translational Frameshifting, Hopping, and Readthrough of Termination Codons , 2000 .

[9]  P. Rigby,et al.  Identification and characterisation of a developmentally regulated mammalian gene that utilises -1 programmed ribosomal frameshifting. , 2001, Nucleic acids research.

[10]  R. Gesteland,et al.  One protein from two open reading frames: mechanism of a 50 nt translational bypass , 2000, The EMBO journal.

[11]  D. Morris,et al.  Programmed translational frameshifting in a gene required for yeast telomere replication , 1997, Current Biology.

[12]  N. Sonenberg,et al.  Translational control of gene expression , 2000 .

[13]  R. Nussinov,et al.  RNA pseudoknots downstream of the frameshift sites of retroviruses , 1991, Genetic Analysis: Biomolecular Engineering.

[14]  J. Rousset,et al.  In vivo HIV-1 frameshifting efficiency is directly related to the stability of the stem-loop stimulatory signal. , 1997, RNA.

[15]  S. Peltz,et al.  Identification of putative programmed -1 ribosomal frameshift signals in large DNA databases. , 1999, Genome research.

[16]  K. Stuart,et al.  Editing of kinetoplastid mitochondrial mRNAs by uridine addition and deletion generates conserved amino acid sequences and AUG initiation codons , 1988, Cell.

[17]  O. Namy,et al.  Translational readthrough of the PDE2 stop codon modulates cAMP levels in Saccharomyces cerevisiae , 2002, Molecular microbiology.

[18]  Y. Takai,et al.  Isolation and characterization of a novel actin filament-binding protein from Saccharomyces cerevisiae , 1998, Oncogene.

[19]  G. C. Roberts,et al.  Alternative splicing: combinatorial output from the genome. , 2002, Current opinion in chemical biology.

[20]  S. Dinesh-Kumar,et al.  A sequence required for -1 ribosomal frameshifting located four kilobases downstream of the frameshift site. , 2001, Journal of molecular biology.

[21]  D. Bedwell,et al.  The efficiency of translation termination is determined by a synergistic interplay between upstream and downstream sequences in Saccharomyces cerevisiae. , 1995, Journal of molecular biology.

[22]  R. Buckingham,et al.  Codon context and protein synthesis: enhancements of the genetic code. , 1994, Biochimie.

[23]  H. Varmus,et al.  Signals for ribosomal frameshifting in the rous sarcoma virus gag-pol region , 1988, Cell.

[24]  I. Stansfield,et al.  Endless possibilities: translation termination and stop codon recognition. , 2001, Microbiology.

[25]  Mark Gerstein,et al.  A small reservoir of disabled ORFs in the yeast genome and its implications for the dynamics of proteome evolution. , 2002, Journal of molecular biology.

[26]  R. Wickner,et al.  [PSI] and [URE3] as yeast prions , 1995, Yeast.

[27]  T. A. Brown,et al.  A rapid and simple method for preparation of RNA from Saccharomyces cerevisiae. , 1990, Nucleic acids research.

[28]  R. Benne,et al.  Major transcript of the frameshifted coxll gene from trypanosome mitochondria contains four nucleotides that are not encoded in the DNA , 1986, Cell.

[29]  H. True,et al.  A yeast prion provides a mechanism for genetic variation and phenotypic diversity , 2000, Nature.

[30]  M. Tuite,et al.  The influence of 5' codon context on translation termination in Saccharomyces cerevisiae. , 1998, European journal of biochemistry.

[31]  R. Wickner.  Prions and RNA viruses of Saccharomyces cerevisiae. , 1996, Annual review of genetics.

[32]  H. Varmus,et al.  Human immunodeficiency virus type 1 gag-pol frameshifting is dependent on downstream mRNA secondary structure: demonstration by expression in vivo , 1992, Journal of virology.

[33]  S. Paushkin,et al.  Propagation of the yeast prion‐like [psi+] determinant is mediated by oligomerization of the SUP35‐encoded polypeptide chain release factor. , 1996, The EMBO journal.

[34]  Michael G. Rosenfeld,et al.  Alternative RNA processing in calcitonin gene expression generates mRNAs encoding different polypeptide products , 1982, Nature.

[35]  L. Klobutcher,et al.  Shifty Ciliates Frequent Programmed Translational Frameshifting in Euplotids , 2002, Cell.

[36]  W. Craigen,et al.  The function, structure and regulation of E. coli peptide chain release factors. , 1987, Biochimie.

[37]  Isabelle Hatin,et al.  Impact of the six nucleotides downstream of the stop codon on translation termination , 2001, EMBO reports.

[38]  J. Rousset,et al.  Versatile vectors to study recoding: conservation of rules between yeast and mammalian cells. , 1995, Nucleic acids research.

[39]  Raymond F. Gesteland,et al.  Computational identification of putative programmed translational frameshift sites , 2002, Bioinform..

[40]  J. F. Atkins,et al.  Analysis of the roles of tRNA structure, ribosomal protein L9, and the bacteriophage T4 gene 60 bypassing signals during ribosome slippage on mRNA. , 2001, Journal of molecular biology.

[41]  J. F. Atkins,et al.  Recoding: dynamic reprogramming of translation. , 1996, Annual review of biochemistry.

[42]  Pavel V Baranov,et al.  Recoding: translational bifurcations in gene expression. , 2002, Gene.

[43]  K. Murata,et al.  Transformation of intact yeast cells treated with alkali cations , 1983 .

[44]  Jean-Pierre Rousset,et al.  UAG readthrough in mammalian cells: Effect of upstream and downstream stop codon contexts reveal different signals , 2001, BMC Molecular Biology.

[45]  J. Piatigorsky,et al.  Alternative RNA splicing of the murine αA-crystallin gene: Protein-coding information within an intron , 1983, Cell.