eIF3h-dependent translational regulation of the AtbZip11 5′ leader. (A) (Top) Schematic of the FLUC reporter coding region fused to original and mutated 5′ leaders of AtbZip11 (590 nt long). Only the four uORFs are drawn to scale (white boxes; 18, 42, 5, and 19 amino acids, respectively). (Middle) Transient expression data from 10-d-old wild-type and eif3h mutant seedlings. The efficiency of translation was calculated as the activity ratio of FLUC versus Renilla luciferase (RLUC) as a co-transformed reference gene. Bars, standard error. (*) P < 0.002 for wild-type (WT) versus eif3h in a two-sided Students t-test. (Bottom) Respective transcript levels of FLUC reporter and translation elongation factor 1α (EF1α) as a control were compared by RT-PCR. (B) Turnover of AtbZip11-FLUC mRNA expressed in stable transgenic plants (Kim et al. 2007) was measured by RT-PCR after blocking transcription with 0.1 mg/mL cordycepin. EF1α served as a control. Neither mRNA was labile over this time course. (C) Translation of the AtbZip11-FLUC reporter construct with the default 3′ UTR from cauliflower mosaic virus was similar to that with the native AtbZip11 3′ UTR. (*) P < 0.002 (n = 5). (D) Undertranslation of the AtbZip11 5′ leader in the eif3h mutant in Arabidopsis protoplasts transformed with in vitro transcripts. “Spacer” is an example for a uORF-less leader that is unaffected by eif3h.
