On the functions of the h subunit of eukaryotic initiation factor 3 in late stages of translation initiation

Abstract

Background: The eukaryotic translation initiation factor 3 (eIF3) has multiple roles during the initiation of translation of cytoplasmic mRNAs. How individual subunits of eIF3 contribute to the translation of specific mRNAs remains poorly understood, however. This is true in particular for those subunits that are not conserved in budding yeast, such as eIF3h.

Results: Working with stable reporter transgenes in Arabidopsis thaliana mutants, it was demonstrated that the h subunit of eIF3 contributes to the efficient translation initiation of mRNAs harboring upstream open reading frames (uORFs) in their 5' leader sequence. uORFs, which can function as devices for translational regulation, are present in over 30% of Arabidopsis mRNAs, and are enriched among mRNAs for transcriptional regulators and protein modifying enzymes. Microarray comparisons of polysome loading in wild-type and eif3h mutant seedlings revealed that eIF3h generally helps to maintain efficient polysome loading of mRNAs harboring multiple uORFs. In addition, however, eIF3h also boosted the polysome loading of mRNAs with long leaders or coding sequences. Moreover, the relative polysome loading of certain functional groups of mRNAs, including ribosomal proteins, was actually increased in the eif3h mutant, suggesting that regulons of translational control can be revealed by mutations in generic translation initiation factors.

Conclusion: The intact eIF3h protein contributes to efficient translation initiation on 5' leader sequences harboring multiple uORFs, although mRNA features independent of uORFs are also implicated.

Figure 1

eIF3h controls the translational efficiency of the AtbZip11 leader in stable, transgenic, reporter gene expression cassettes. (a) Schematic of the reporter gene T-DNA structure. The efficiency of translation initiation on a given 5' leader sequence is measured by comparing the activity of the associated firefly (Fluc) reporter gene with the activity of the Renilla luciferase (Rluc) reference gene, which is expressed under the control of the cauliflower mosaic virus 35S promoter (35S) and the generic 5' leader sequence from tobacco etch virus (TL). (b) Translational efficiency of the AtbZip11 (ATB2) leader in wild-type (WT) and eif3h mutant seedlings. Seedlings were germinated for nine days on solid agar medium in the light. The figure shows raw Fluc/Rluc activity ratios from seven individual experiments conducted with one transgenic line. The data are representative of other raw data that underlie Figure 1c-e and Figure 2. (c) Translational efficiency of the AtbZip11 leader in wild-type (WT) and eif3h mutant seedlings. All six independent transgenic lines examined are shown. The bars indicate Fluc/Rluc ratios (left y-axis), while the triangles show the ratio of translational efficiency between wild-type (Wt) and mutant plants (right y-axis). The Wt/eif3h bracket between 0.5 and 1.5 is highlighted in gray to facilitate comparison between panels. SE, standard error. (d) Translational efficiency of the tobacco etch virus leader (TL) in wild-type (Wt) and eif3h mutant seedlings. Data from five lines are displayed as for (c). (e) Translational efficiency of the leader of the Arabidopsis LHY (At1g01060) gene. Fluc/Rluc bars for line 7 are displayed at 10% of the original values.

Figure 2

uORF2b contributes to poor translatability of the AtbZip11 leader in the eif3h mutant. (a) Schematic of the arrangement of uORFs in the AtbZip11 leader. uORF2b was mutated by changing its start codon into a stop codon. (b) Translation efficiencies of the AtbZip11 leader lacking uORF2b in three different organs of two-week-old seedlings. The number of lines examined is indicated (n), as are p values derived from pairwise t-tests. For details see legend to Figure 1. (c) Summary of transgenic reporter gene translation assays on six different leader sequences. The number of transgenic lines examined is indicated for each leader, as is the number of uORFs per leader. The letters a and b indicate homogeneous subsets as determined by ANOVA/Tukey test. Thus, leaders that do not share the same letter (a, b) differ significantly in their dependence on the eIF3h protein. SE, standard error. (d) Reverse-transcriptase PCR analysis of FLUC mRNA levels in representative transgenic lines harboring TL-FLUC, AtbZip11-FLUC or AtbZip11/2b-FLUC transgenes. The EF1α mRNA was analyzed as a control for equal mRNA levels. The ethidium-bromide stained gels shown here are consistent with other repeat experiments performed with other subsaturating numbers of PCR cycles.

Figure 3

Microarray analysis of polysome loading in the eif3h mutant. (a) Experimental design for the isolation of polysomal (PL) and non-polysomal (NP) RNAs. After sucrose density gradient centrifugation, samples were collected into 12 fractions. The integrity of the density gradient was confirmed by agarose gel electrophoresis and visualization of ribosomal RNAs with ethidium bromide. Microarray probes were generated from pooled samples as indicated. (b) Log₂ transformed average translation states (TL = PL/NP) of the eif3h mutant were plotted against the data from wild-type plants. (c) Effects on polysome loading by the eif3h mutation (Log₂ [TL]_3h/[TL]_WT) were not generally correlated with effects on transcript levels (Log₂ [TC]_3h/[TC]_WT). An arbitrary two-fold cut-off was applied to highlight responsive genes (dotted lines). The number of genes affected both transcriptionally and translationally is very small (25 out of 6,238 genes for which reproducible data were available). Among them, the eIF3h mRNA is indicated by an arrow head.

Figure 4

Survey of trends in translational stimulation and repression among functional classes of genes. The changes in (a) translation states or (b) transcript level observed between wild type and eif3h are shown after gene ontology analysis using MapMan v1.8.0 [33]. Bars represent the percentage of responsive genes in a particular class when a two-fold cut-off was applied. X² tests were carried out to evaluate the extent of deviation from the average pattern and p values are given.

Figure 5

Certain functional classes of mRNAs show a coordinated translational response to the eif3h mutation. Microarray data were plotted onto Arabidopsis biochemical pathways and functional categories using MapMan v1.8.0. Each square represents a single gene. On the log color scale, light blue refers to a 2-fold (log₂ = 1) stimulation of polysome loading or transcript level in the eif3h mutant compared to wild type. Note the translational stimulation of ribosomal proteins and plastid proteins in the eif3h mutant and the translational reduction for receptor kinases, transcription factors, F-box proteins, and protein modifying enzymes. Other classes are shown as non-significant controls.

Figure 6

Characterization of Arabidopsis 5' leader sequences. The analysis is based on a set of sequences obtained from cap-purified mRNAs (see Materials and methods). (a) Length distribution. (b) Number of uAUGs. (c) Correlation between length of the leader and number of uAUGs. (d) Distribution of uORF lengths among the 12,129 bona fide full-length leader sequences. uORFs that overlap the main ORF were not included in this analysis. (e) The frequency of each dinucleotide (AA, AC, and so on) was determined empiricially across all 5' leaders (not shown). Then, the theoretical frequency of each triplet was predicted based on the dinucleotide data (see Materials and methods for details) and set to 100%. The empirical frequency of each triplet across all 5' leaders was then expressed in relation to the predicted frequency.

Figure 7

eIF3h is responsible for enhancing the translation state of mRNAs harboring upstream AUGs. mRNAs were classified into bins according to the difference in translation state between eif3h mutant and wild type (log₂ [TL]_3h/[TL]_WT). The proportion of genes containing uAUGs was determined for each bin. 'Strong context' refers to uAUGs in the sequence context AnnAUGn or GnnAUGG, which are close matches to the optimal Kozak consensus for translation initiation.

Figure 8

uORF content and eIF3h-dependent polysome loading are correlated across functional categories of genes. Each datapoint represents a functional class of genes. The percentages of translationally up- or downregulated genes were plotted against the percentages of uAUG-containing genes in a given functional class. The four classes with the highest occurrence of uORFs and the two classes with the lowest occurrence are highlighted with pink and green circles, respectively. The left panels (a, c) focus on genes whose polysome loading is reduced in the eif3h mutant; whereas the right panels (b, d) focus on genes with stimulation of polysome loading. (a, b) Stringent cutoff; only genes with a two-fold or higher difference in polysome loading were considered. (c, d) Relaxed cutoff; genes with a 1.3-fold or higher difference in polysome loading were considered. Note that the correlation is apparent in each case.

Figure 9

Identification of structural parameters that predict mRNA polysome loading in wild type and eif3h mutants. The average translation state of mRNAs was plotted separately for wild-type and eif3h mutant plants after subdividing the transcriptome (5,101 genes with polysome loading data and full-length cDNA support) according to the following parameters: (a) number of uAUGs in the 5' leader; (b) the eif3h mutation did not cause a global reduction in mRNA transcript levels for uORF-containing mRNAs; (c) length of the 5' leader in nucleotides (nt); (d) similar to (c), but leaders were classified into subgroups according to the number of uORFs. For clarity, we plotted only the difference in translation state between eif3h and wild type (colored lines). The stippled line denotes the percentage of leaders in each length class (cumulative). (e) Length of the 3' untranslated region. (f) Length of the protein-coding region of the main ORF. Standard errors are shown. The asterisk indicates a significant difference between the translation state in wild type and eif3h mutant (p < 0.05) according to a t-test (unpaired, two-tail).

Figure 10

Contribution of the length of the main ORF versus the presence of uORFs to the translation state of mRNAs. Bins selected according to the extent of eIF3h-dependence (Figure 7) were examined for the percentage of genes with a long (> 1,300 nt) main ORF but no uAUG, a short main ORF (< 1,300 nt) and no uAUG, and any number of uAUGs.