mRNA sequence features that contribute to translational regulation in Arabidopsis

Abstract

DNA microarrays were used to evaluate the regulation of the proportion of individual mRNA species in polysomal complexes in leaves of Arabidopsis thaliana under control growth conditions and following a mild dehydration stress (DS). The analysis determined that the percentage of an individual gene transcript in polysomes (ribosome loading) ranged from over 95 to <5%. DS caused a decrease in ribosome loading from 82 to 72%, with maintained polysome association for over 60% of the mRNAs with an increased abundance. To identify sequence features responsible for translational regulation, ribosome loading values and features of full-length mRNA sequences were compared. mRNAs with extreme length or high GU content in the 5'-untranslated regions (5'-UTRs) were generally poorly translated. Under DS, mRNAs with both a high GC content in the 5'-UTR and long open reading frame showed a significant impairment in ribosome loading. Evaluation of initiation A+1UG codon context revealed distinctions in the frequency of adenine in nucleotides -10 to -1 (especially at -4 and -3) in mRNAs with different ribosome loading values. Notably, the mRNA features that contribute to translational regulation could not fully explain the variation in ribosome loading, indicating that additional factors contribute to translational regulation in Arabidopsis.

Figure 1

Extensive variability in ribosome loading under NS conditions and global reduction in ribosome loading in response to DS. nRL values were determined for the transcripts detected above background in both non-polysomal and polysomal RNA fractions under at least one condition by DNA microarray hybridization (7). The nRL value for each mRNA was plotted as the percentage of the individual mRNA species in polysomes under NS and DS conditions. Arrows indicate the positions and values of the average nRL (determined with log₂-transformed values) for the NS (average log₂nRL 2.15 ± 0.87 for n = 11 746) and DS (average log₂nRL = 1.3 ± 0.88 for n = 12 310). Averages were statistically different as determined by the Student's t-test on the log₂nRL values (P < 10⁻⁴).

Figure 2

Effect of mRNA 5′-UTR, coding region and 3′-UTR length on ribosome loading. FL-cDNA sequences were grouped based on the length of 5′-UTR, length of coding ORF and 3′-UTR with a window of 25 nt (5′-UTR), 500 nt (ORF) and 40 nt (3′-UTR). The average length of each region within each group was plotted against the average ribosome loading (percentage of an individual mRNA in the polysomes) under NS (closed diamond) or DS (open square). mRNAs that were detected in non-polysomal and polysomal fractions under at least one of the conditions were used for the analysis (A) 5′-UTR, n = 2724, NS; n = 2845, DS; (B) ORF, n = 10 628, for NS; n = 11 056, for DS; and (C) 3′-UTR, n = 9018, for NS; n = 9405, for DS. Error bars indicate S.E. Statistical significance of ribosome loading in each range was determined for log₂nRL values against the average log₂nRL value for each condition by the Student's t-test (^*, P < 0.05, ^**, P < 0.01, ^***, P < 0.001).

Figure 3

Effect of potential RNA secondary structure formation and GC content in the 5′-UTR on ribosome loading. mRNAs were grouped based on the predicted free energy (ΔG kcal/mol) or the GC content with a 15 kcal/mol (ΔG) or 5% (GC content) window, respectively. (A) The average predicted free energy of the 5′-UTRs in each group under NS (n = 2641, closed diamond) and DS (n = 2758, open square) was plotted. (B) Effect of 5′-UTR secondary structure for the mRNA with 5′-UTR length of 80–180 nt. mRNA sequences from FL-cDNA are grouped based on the predicted free energy (ΔG kcal/mol). (C) The average GC content of the 5′-UTR of each group under NS (closed diamond) and DS (open square) was plotted. (D) The average change in ribosome loading in response to DS was plotted against the average change in GC content of the 5′-UTR of each group in response to DS. Statistical significance of ribosome loading or change in ribosome loading was determined against the average value under each condition by Student's t-test (^*, P < 0.05, ^**, P < 0.01, ^***, P < 0.001). Error bars indicate S.E.

Figure 4

Effect of presence of an AUG or ORF upstream of initiation codon on ribosome loading. mRNAs in FL-cDNA sequence dataset with or without an uAUG (an AUG upstream of the initiation codon) were selected. (A) Frequency distribution of uORF length. (B) Ribosome loading values under NS and DS conditions were compared for mRNAs that lacked (black bar) or possessed one or more uAUG(s) (white bar). Statistical significance of ribosome loading values (log₂nRL) of mRNAs with an uAUG as compared with an average log₂nRL of mRNAs without uAUG was determined under NS or DS conditions by Student's t-test (^***P < 0.0001). Error bars indicate S.E. Values above bars indicate the sample size. (C) Ribosome loading values under NS and DS conditions for mRNAs with a uORF of varying length.

Figure 5

Evaluation of frequency of nucleotides surrounding the initiation codon AUG in mRNAs. TIGR cDNA sequences with 10 or greater nucleotides were selected for the determination of nucleotide frequency at positions between −10 and +5 (initiation codon AUG corresponds to position +1 to +3). (A) All genes with mRNAs detected under NS and DS conditions (n = 7870). mRNAs with very high (approximately highest 1% ribosome loading values) (B and D) and very low (approximately lowest 5% ribosome loading values) nRL (C and E) under NS and DS conditions, respectively. The statistical significance of the frequency of the nucleotide with the highest value at each position was determined by Chi-square test against the combined frequency of other nucleotides of all mRNAs under the same condition; asterisks in parenthesis indicate the comparison against mRNAs with very low nRL, ^*, P < 0.05, ^**, P < 0.01, ^***, P < 0.001. Gene numbers (n) in samples indicated.

Figure 6

Effect of most frequent nucleotide at position −10 to +5 on dehydration-induced change in ribosome loading. Difference between average ribosome loading values of mRNAs with and without the most frequent nucleotide determined from the mRNA set with highest 1% ribosome loading under NS and DS at each position between −10 and +5. Statistical significance of the difference was determined by Student's t-test for the average log₂nRL value of mRNAs with and without the corresponding nucleotide (^*, P < 0.05, ^**, P < 0.01, ^***, P < 0.001).