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Nucleic Acids Res. 2017 Sep 6;45(15):8844-8858. doi: 10.1093/nar/gkx528.

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

Author information

1
Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan.
2
Graduate School of Horticulture, Chiba University, Chiba 263-8522, Japan.
3
Graduate School of Life Science, Hokkaido University, Sapporo 060-0810, Japan.

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.

PMID:
28637336
PMCID:
PMC5587730
DOI:
10.1093/nar/gkx528
[Indexed for MEDLINE]
Free PMC Article

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