Identification of Arabidopsis thaliana upstream open reading frames encoding peptide sequences that cause ribosomal arrest

Abstract Specific sequences of certain nascent peptides cause programmed ribosomal arrest during mRNA translation to control gene expression. In eukaryotes, most known regulatory arrest peptides are encoded by upstream open reading frames (uORFs) present in the 5′-untranslated region of mRNAs. However, to date, a limited number of eukaryotic uORFs encoding arrest peptides have been reported. Here, we searched for arrest peptide-encoding uORFs among Arabidopsis thaliana uORFs with evolutionarily conserved peptide sequences. Analysis of in vitro translation products of 22 conserved uORFs identified three novel uORFs causing ribosomal arrest in a peptide sequence-dependent manner. Stop codon-scanning mutagenesis, in which the effect of changing the uORF stop codon position on the ribosomal arrest was examined, and toeprint analysis revealed that two of the three uORFs cause ribosomal arrest during translation elongation, whereas the other one causes ribosomal arrest during translation termination. Transient expression assays showed that the newly identified arrest-causing uORFs exerted a strong sequence-dependent repressive effect on the expression of the downstream reporter gene in A. thaliana protoplasts. These results suggest that the peptide sequences of the three uORFs identified in this study cause ribosomal arrest in the uORFs, thereby repressing the expression of proteins encoded by the main ORFs.

[1]  Daniel N. Wilson,et al.  A combined cryo-EM and molecular dynamics approach reveals the mechanism of ErmBL-mediated translation arrest , 2016, Nature Communications.

[2]  M. Sachs,et al.  Arginine changes the conformation of the arginine attenuator peptide relative to the ribosome tunnel. , 2012, Journal of molecular biology.

[3]  T. Berberich,et al.  An inhibitory effect of the sequence-conserved upstream open-reading frame on the translation of the main open-reading frame of HsfB1 transcripts in Arabidopsis. , 2012, Plant, cell & environment.

[4]  M. Sachs,et al.  Ribosome Elongation Stall Directs Gene-specific Translation in the Integrated Stress Response* , 2016, The Journal of Biological Chemistry.

[5]  D. Morris,et al.  Polyamine regulation of ribosome pausing at the upstream open reading frame of S-adenosylmethionine decarboxylase. , 2001, The Journal of biological chemistry.

[6]  T. Martin Schmeing,et al.  An induced-fit mechanism to promote peptide bond formation and exclude hydrolysis of peptidyl-tRNA , 2005, Nature.

[7]  H. Bernstein,et al.  The plasticity of a translation arrest motif yields insights into nascent polypeptide recognition inside the ribosome tunnel. , 2009, Molecular cell.

[8]  C. Yanofsky,et al.  Changes produced by bound tryptophan in the ribosome peptidyl transferase center in response to TnaC, a nascent leader peptide. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[9]  Mark L Crowe,et al.  Evidence for conservation and selection of upstream open reading frames suggests probable encoding of bioactive peptides , 2006, BMC Genomics.

[10]  R. Dohmen,et al.  Polyamine sensing by nascent ornithine decarboxylase antizyme stimulates decoding of its mRNA , 2011, Nature.

[11]  Karolina M. Pajerowska-Mukhtar,et al.  The HSF-like Transcription Factor TBF1 Is a Major Molecular Switch for Plant Growth-to-Defense Transition , 2012, Current Biology.

[12]  S. Naito,et al.  Sucrose sensing through nascent peptide‐meditated ribosome stalling at the stop codon of Arabidopsis bZIP11 uORF2 , 2017, FEBS letters.

[13]  Daniel N. Wilson,et al.  Molecular basis for erythromycin-dependent ribosome stalling during translation of the ErmBL leader peptide , 2014, Nature Communications.

[14]  M. Mayer,et al.  A Dual Upstream Open Reading Frame-based Autoregulatory Circuit Controlling Polyamine-responsive Translation* , 2005, Journal of Biological Chemistry.

[15]  V. Mootha,et al.  Upstream open reading frames cause widespread reduction of protein expression and are polymorphic among humans , 2009, Proceedings of the National Academy of Sciences.

[16]  D. Morris,et al.  Upstream Open Reading Frames as Regulators of mRNA Translation , 2000, Molecular and Cellular Biology.

[17]  J. Bailey-Serres,et al.  mRNA sequence features that contribute to translational regulation in Arabidopsis , 2005, Nucleic acids research.

[18]  Koreaki Ito,et al.  Multisite ribosomal stalling: a unique mode of regulatory nascent chain action revealed for MifM. , 2012, Molecular cell.

[19]  Emily R. Gordon,et al.  Interactions of the TnaC nascent peptide with rRNA in the exit tunnel enable the ribosome to respond to free tryptophan , 2013, Nucleic acids research.

[20]  M. Sachs,et al.  Toeprint analysis of the positioning of translation apparatus components at initiation and termination codons of fungal mRNAs. , 2002, Methods.

[21]  Y. Hanzawa,et al.  The dwarf phenotype of the Arabidopsis acl5 mutant is suppressed by a mutation in an upstream ORF of a bHLH gene , 2006, Development.

[22]  S. Naito,et al.  An upstream open reading frame represses expression of a tomato homologue of Arabidopsis ANAC096 , a NAC domain transcription factor gene, in a peptide sequence-dependent manner , 2015 .

[23]  Z. Wang,et al.  A Highly Conserved Mechanism of Regulated Ribosome Stalling Mediated by Fungal Arginine Attenuator Peptides That Appears Independent of the Charging Status of Arginyl-tRNAs* , 1999, The Journal of Biological Chemistry.

[24]  Koreaki Ito,et al.  The Ribosomal Exit Tunnel Functions as a Discriminating Gate , 2002, Cell.

[25]  Nobutaka Mitsuda,et al.  Arabidopsis HsfB1 and HsfB2b Act as Repressors of the Expression of Heat-Inducible Hsfs But Positively Regulate the Acquired Thermotolerance1[C][W][OA] , 2011, Plant Physiology.

[26]  Daniel N. Wilson,et al.  Drug sensing by the ribosome induces translational arrest via active site perturbation. , 2014, Molecular cell.

[27]  S. Naito,et al.  Nascent peptide-mediated translation elongation arrest coupled with mRNA degradation in the CGS1 gene of Arabidopsis. , 2005, Genes & development.

[28]  A. Geballe,et al.  Coding sequence-dependent ribosomal arrest at termination of translation , 1996, Molecular and cellular biology.

[29]  W. Busch,et al.  Heat shock factors HsfB1 and HsfB2b are involved in the regulation of Pdf1.2 expression and pathogen resistance in Arabidopsis. , 2009, Molecular plant.

[30]  K. Ito,et al.  Secretion monitor, SecM, undergoes self-translation arrest in the cytosol. , 2001, Molecular cell.

[31]  A. Rich,et al.  Partial resistance of nascent polypeptide chains to proteolytic digestion due to ribosomal shielding. , 1967, Journal of molecular biology.

[32]  Koreaki Ito,et al.  Arrest peptides: cis-acting modulators of translation. , 2013, Annual review of biochemistry.

[33]  M. Hirai,et al.  The Minimum Open Reading Frame, AUG-Stop, Induces Boron-Dependent Ribosome Stalling and mRNA Degradation , 2016, Plant Cell.

[34]  Hiro Takahashi,et al.  BAIUCAS: a novel BLAST-based algorithm for the identification of upstream open reading frames with conserved amino acid sequences and its application to the Arabidopsis thaliana genome , 2012, Bioinform..

[35]  M. Kozak,et al.  Recognition of AUG and alternative initiator codons is augmented by G in position +4 but is not generally affected by the nucleotides in positions +5 and +6 , 1997, The EMBO journal.

[36]  A. Geballe,et al.  Inhibition of nascent-peptide release at translation termination , 1996, Molecular and cellular biology.

[37]  R. Jorgensen,et al.  Conserved Peptide Upstream Open Reading Frames are Associated with Regulatory Genes in Angiosperms , 2012, Front. Plant Sci..

[38]  Daniel N. Wilson,et al.  Structure of the Bacillus subtilis 70S ribosome reveals the basis for species-specific stalling , 2015, Nature Communications.

[39]  Anika Wiese-Klinkenberg,et al.  Sucrose Control of Translation Mediated by an Upstream Open Reading Frame-Encoded Peptide1[W][OA] , 2009, Plant Physiology.

[40]  Koreaki Ito,et al.  Genetically encoded but nonpolypeptide prolyl-tRNA functions in the A site for SecM-mediated ribosomal stall. , 2006, Molecular cell.

[41]  D. Morris,et al.  The Upstream Open Reading Frame of the mRNA Encoding S-Adenosylmethionine Decarboxylase Is a Polyamine-responsive Translational Control Element* , 1996, The Journal of Biological Chemistry.

[42]  K. Pogliano,et al.  A ribosome–nascent chain sensor of membrane protein biogenesis in Bacillus subtilis , 2009, The EMBO journal.

[43]  S. Ho,et al.  Site-directed mutagenesis by overlap extension using the polymerase chain reaction. , 1989, Gene.

[44]  Giovanni Bosco,et al.  Comparative genomic analysis of novel conserved peptide upstream open reading frames in Drosophila melanogaster and other dipteran species , 2008, BMC Genomics.

[45]  Justin N Vaughn,et al.  Known and novel post-transcriptional regulatory sequences are conserved across plant families. , 2012, RNA.

[46]  H. Stark,et al.  Spontaneous reverse movement of mRNA-bound tRNA through the ribosome , 2007, Nature Structural &Molecular Biology.

[47]  T. Fujiwara,et al.  Ribosomes in a Stacked Array , 2014, The Journal of Biological Chemistry.

[48]  C. Yanofsky,et al.  Nascent polypeptide sequences that influence ribosome function. , 2011, Current opinion in microbiology.

[49]  M. Sachs,et al.  The Arginine Attenuator Peptide Interferes with the Ribosome Peptidyl Transferase Center , 2012, Molecular and Cellular Biology.

[50]  L. Gold,et al.  Extension inhibition analysis of translation initiation complexes. , 1988, Methods in enzymology.

[51]  G. Blobel,et al.  Cell-free translation of messenger RNA in a wheat germ system. , 1983, Methods in enzymology.

[52]  G. Mize,et al.  In Vitro Translation of the Upstream Open Reading Frame in the Mammalian mRNA EncodingS-Adenosylmethionine Decarboxylase* , 2000, The Journal of Biological Chemistry.

[53]  S. Kanaya,et al.  Effect of the sequence context of the AUG initiation codon on the rate of translation in dicotyledonous and monocotyledonous plant cells. , 2010, Journal of bioscience and bioengineering.

[54]  W. Merrick,et al.  Analysis of 40 S and 80 S complexes with mRNA as measured by sucrose density gradients and primer extension inhibition. , 1992, The Journal of biological chemistry.

[55]  J. Lingner,et al.  AUF1/HnRNP D RNA binding protein functions in telomere maintenance. , 2012, Molecular cell.

[56]  C. Yanofsky,et al.  The mechanism of tryptophan induction of tryptophanase operon expression: Tryptophan inhibits release factor-mediated cleavage of TnaC-peptidyl-tRNAPro , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[57]  M. Kozak.  Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes , 1986, Cell.

[58]  L. Gold,et al.  [27] Extension inhibition analysis of translation initiation complexes☆ , 1988 .

[59]  S. Naito,et al.  Polyamine-responsive ribosomal arrest at the stop codon of an upstream open reading frame of the AdoMetDC1 gene triggers nonsense-mediated mRNA decay in Arabidopsis thaliana. , 2014, Plant & cell physiology.

[60]  C. Yanofsky,et al.  Features of ribosome-peptidyl-tRNA interactions essential for tryptophan induction of tna operon expression. , 2005, Molecular cell.

[61]  M. Menges,et al.  Synchronous Arabidopsis suspension cultures for analysis of cell-cycle gene activity. , 2002, The Plant journal : for cell and molecular biology.

[62]  L. Lindahl,et al.  Effects on Translation Pausing of Alterations in Protein and RNA Components of the Ribosome Exit Tunnel , 2008, Journal of bacteriology.

[63]  D. Sabatini,et al.  CONTROLLED PROTEOLYSIS OF NASCENT POLYPEPTIDES IN RAT LIVER CELL FRACTIONS , 1970, The Journal of cell biology.

[64]  A. Mankin,et al.  Molecular mechanism of drug-dependent ribosome stalling. , 2008, Molecular cell.

[65]  H. Ramu,et al.  Programmed drug‐dependent ribosome stalling , 2009, Molecular microbiology.

[66]  W. Gray,et al.  SAUR Proteins as Effectors of Hormonal and Environmental Signals in Plant Growth. , 2015, Molecular plant.

[67]  R. Wek,et al.  Phosphorylation of eIF2 Facilitates Ribosomal Bypass of an Inhibitory Upstream ORF to Enhance CHOP Translation*♦ , 2011, The Journal of Biological Chemistry.

[68]  M. Sachs,et al.  Crucial elements that maintain the interactions between the regulatory TnaC peptide and the ribosome exit tunnel responsible for Trp inhibition of ribosome function , 2011, Nucleic acids research.

[69]  Daniel N. Wilson,et al.  Structural basis for translational stalling by human cytomegalovirus and fungal arginine attenuator peptide. , 2010, Molecular cell.

[70]  P. S. Lovett,et al.  Ribosome regulation by the nascent peptide. , 1996, Microbiological reviews.

[71]  U. Conrath,et al.  Heat Shock Factor HsfB1 Primes Gene Transcription and Systemic Acquired Resistance in Arabidopsis1[W] , 2012, Plant Physiology.

[72]  Masayuki Ishikawa,et al.  Arabidopsis cell-free extract, ACE, a new in vitro translation system derived from Arabidopsis callus cultures. , 2011, Plant & cell physiology.

[73]  Celine A. Hayden,et al.  Identification of novel conserved peptide uORF homology groups in Arabidopsis and rice reveals ancient eukaryotic origin of select groups and preferential association with transcription factor-encoding genes , 2007, BMC Biology.

[74]  Satoshi Naito,et al.  Identification of novel Arabidopsis thaliana upstream open reading frames that control expression of the main coding sequences in a peptide sequence-dependent manner , 2015, Nucleic acids research.

[75]  Jennifer L. Nemhauser,et al.  Different Plant Hormones Regulate Similar Processes through Largely Nonoverlapping Transcriptional Responses , 2006, Cell.

[76]  D. Klepacki,et al.  Nascent peptide in the ribosome exit tunnel affects functional properties of the A-site of the peptidyl transferase center. , 2011, Molecular cell.