Upstream Open Reading Frames (uORF) Analysis Of Plant MRNAs And Gene TaPSG719

Upstream open reading frames (uORF) in 5'-untranslated regions (5'-UTR) of eukaryotic mRNAs play a important role in translation efficiency. Computer analysis of upstream ATG and uORF of 5'-untranslated regions (5'-UTR) of plants mRNA adopted from the nucleotide sequence databank was carried out. Statistical analysis has revealed that up to 18% of 5'- UTRs contain uATG which is much more than the earlier estimate. Among them, about 50% of the genes have two uATG and nearly 20% of them have two uATG. About 85% of uORFs are nonoverlapping. 30% of uORF peptide is 1-5 aa,. and about 80% of uORF fall in the range of below 20 aa. Sequences flanking the uATG codon differs strikingly from the fuctional initiation codons and the upstream ATG triplets are more frequently located in a nonoptimal context. Consenses sequences of the ATG codon contexts of mRNA with and without uATG is similar but the ATG codon contexts of mRNA without uATG are more frequently located in a optimal context than that of mRNA with uATG. Most of mRNA with uATG are possibly relation with regulatory function. In addition, Most of uORFs have no similiarity between plant mRNA species whereas sequences of a few uORFs of are highly conserved, for example, uORF of 5-Adenosyl-L-methionine decarboxylase (AdoMetDC) are 75-100% homology between plant mRNA species, which much more conserved than AdoMetDC.TaPSG719, GenBank: AY451238 TaPSG719 clone is 1,172-bp long, and encodes a protein of 188 amino acids long with a pi of 12.1. Southern blot indicated that TaPSG719 was a member of a small family. Northern blot and RT-PCR analysis indicated that TaPSG719 transcripts showed specific expression of transcript inmature pollen, and undetectable signals in microspore, immature seed, stem, young leave, root and ovary. During pollen development, TaPSG719 transcripts were first detectable at 5 days before anthesis and increased rapidly at middle stages of pollen development with maximum levels at 4 days before anthesis, and decrease during pollen maturation.To investigate the role uORF on translation, in vitro transcription/translation reactions with various constructs of deletion and mutation were performed using wheat germ extract. The uORF affected positively downstream translation in wheat germ extract.