The upstream open reading frame of the Arabidopsis AtMHX gene has a strong impact on transcript accumulation through the nonsense-mediated mRNA decay pathway.
Approximately 20% of plant genes possess upstream open-reading frames (uORFs). The effect of uORFs on gene expression has mainly been studied at the translational level. Very little is known about the impact of plant uORFs on transcript content through the nonsense-mediated mRNA decay (NMD) pathway, which degrades transcripts bearing premature termination codons (PTCs). Here we examine the impact of the uORF of the Arabidopsis AtMHX gene on transcript accumulation. The suggestion that this uORF exposes transcripts containing it to NMD is supported by (i) the increase in transcript levels upon eliminating the uORF from constructs containing it, (ii) experiments with a modified uORF-peptide, which excluded peptide-specific degradation mechanisms, (iii) the increase in levels of the native AtMHX transcript upon treatment with cycloheximide, which inhibits translation and blocks NMD, and (iv) the sensitivity of transcripts containing the uORF of AtMHX to the presence of introns. We also showed that introns can increase NMD efficiency not only in transcripts having relatively short 3' untranslated regions (UTRs), but also in uORF-containing transcripts. AtMHX transcript levels were almost unaltered in mutants of the NMD factors UPF3 and UPF1. Possible reasons, including the existence of a NMD-compensatory mechanism, are discussed. Interestingly, the levels of UPF3 transcript were higher in upf1 mutants, suggesting a compensatory mechanism that links weak function of the NMD machinery to increased expression of UPF3. Our findings highlight that uORFs, which are abundant in plants, can not only inhibit translation but also strongly affect transcript accumulation.
Saul
Helen
H
The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel.
Elharrar
Einat
E
Gaash
Rachel
R
Eliaz
Dror
D
Valenci
Meital
M
Akua
Tsofit
T
Avramov
Meital
M
Frankel
Neta
N
Berezin
Irina
I
Gottlieb
Dror
D
Elazar
Meirav
M
David-Assael
Ora
O
Tcherkas
Vered
V
Mizrachi
Keren
K
Shaul
Orit
O
D020413:3' Untranslated Regions; D017920:Antiporters / Q000235:genetics* / Q000378:metabolism; D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics* / Q000378:metabolism; D018389:Codon, Nonsense; D003513:Cycloheximide / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics; D018506:Gene Expression Regulation, Plant; D017930:Genes, Reporter; D007438:Introns; D016254:Mutagenesis, Insertional; D016366:Open Reading Frames; D020871:RNA Stability; D018749:RNA, Plant / Q000235:genetics / Q000378:metabolism
D020413:3' Untranslated Regions; D017920:Antiporters; D029681:Arabidopsis Proteins; C120847:AtMHX protein, Arabidopsis; D018389:Codon, Nonsense; D004269:DNA, Bacterial; D018749:RNA, Plant; C036483:T-DNA; C504799:UPF3 protein, Arabidopsis; D003513:Cycloheximide