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. 2014 Jan 7;111(1):E203-12.
doi: 10.1073/pnas.1317811111. Epub 2013 Dec 23.

Translational dynamics revealed by genome-wide profiling of ribosome footprints in Arabidopsis

Piyada Juntawong  1 , Thomas Girke, Jérémie Bazin, Julia Bailey-Serres
Affiliations

Translational dynamics revealed by genome-wide profiling of ribosome footprints in Arabidopsis

Piyada Juntawong et al. Proc Natl Acad Sci U S A. .

Abstract

Translational regulation contributes to plasticity in metabolism and growth that enables plants to survive in a dynamic environment. Here, we used the precise mapping of ribosome footprints (RFs) on mRNAs to investigate translational regulation under control and sublethal hypoxia stress conditions in seedlings of Arabidopsis thaliana. Ribosomes were obtained by differential centrifugation or immunopurification and were digested with RNase I to generate footprint fragments that were deep-sequenced. Comparison of RF number and position on genic regions with fragmented total and polysomal mRNA illuminated numerous aspects of posttranscriptional and translational control under both growth conditions. When seedlings were oxygen-deprived, the frequency of ribosomes at the start codon was reduced, consistent with a global decline in initiation of translation. Hypoxia-up-regulated gene transcripts increased in polysome complexes during the stress, but the number of ribosomes per transcript relative to normoxic conditions was not enhanced. On the other hand, many mRNAs with limited change in steady-state abundance had significantly fewer ribosomes but with an overall similar distribution under hypoxia, consistent with restriction of initiation rather than elongation of translation. RF profiling also exposed the inhibitory effect of upstream ORFs on the translation of downstream protein-coding regions under normoxia, which was further modulated by hypoxia. The data document translation of alternatively spliced mRNAs and expose ribosome association with some noncoding RNAs. Altogether, we present an experimental approach that illuminates prevalent and nuanced regulation of protein synthesis under optimal and energy-limiting conditions.

Keywords: alternative splicing; long intergenic noncoding RNA; ribosome profiling; translational efficiency; uORF.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
RF profiling analysis of control (normoxic) and hypoxic seedlings exposes regulation of translational initiation. (A) Overview of experimental strategy. (B) Absorbance (A254 nm) of sucrose density gradient fractionated ribosomes from RNase I- or mock-treated (control) cell extracts. (C) Representative sucrose density gradient profiles of ribosomes from normoxic and 2-h hypoxic seedlings. (D) Size distribution and relative abundance of RF reads and total and polysomal mRNA processed in parallel. (E) Fraction of 30-nt reads at the first (A), second (U), and third (G) nucleotide positions, respectively, of the start codon [average and SD for nonstress samples (RF#1 and RF#2)]. (F) Comparison of RF rpkM values per protein-coding ORF for all genes with >128 reads from one representative biological replicate. (G) Coverage values of 30-nt reads within and at the ends of the ORFs of genes. Inferred ribosome position relative to the acceptor (A), peptidyl (P), and exit (E) sites of the ribosome with the start codon in the P site and the stop codon in the A site. Bars show RFs, and lines show total (T) and polysomal (P) mRNA reads for the two conditions. Reads are mapped at the first nucleotide of the RF fragment. Data in D–G represent reads mapped uniquely to the genome with two or fewer mismatches. D, F, and G are data from RF#1. Figs. S2 and S3 provide corresponding analyses of other datasets.
Fig. 2.
Fig. 2.
Translational efficiency of individual mRNAs is perturbed by hypoxia. (A) Comparison of translational efficiency under normoxia and hypoxia (reads mapped to ORFs with ≥5 rpkM). Values are given for the number of genes evaluated and genes with conditionally altered translational efficiency. (B) Change in mRNA abundance and translational efficiency in response to hypoxia for the same genes as in A. (C) Coverage of RF and TRAP-RF, polysomal mRNA, and total mRNA reads on selected genes. ADH1 (ALCOHOL DEHYDROGENASE1; AT1G77120) and LBD41 (LATERAL ORGAN BOUNDARY DOMAIN41; AT3G02550) are core hypoxia-responsive genes (2). RPL36aA (AT3G23390) and RPL10C (AT1G66580) encode ribosomal proteins. TPPA (TREHALOSE PHOSPHATE PHOSPHATASE A; AT5G51460) has two transcription start sites with use of the 5′ site more prevalent under hypoxia. The red arrowheads indicate a region present only in the shorter transcript. The black arrows indicate the two transcription start sites. PERK10 (PROLINE-RICH EXTENSIN-LIKE RECEPTOR KINASE 10; AT1G26150) shows higher ribosome occupancy under hypoxia. The maximum read value of the scale used for each gene is indicated at the upper left.
Fig. 3.
Fig. 3.
Hypoxia alters translation of uORFs relative to mORFs on multicistronic mRNAs. (A) Coverage values of 30-nt reads at the annotated start codon region, uORF coding sequence, and stop codon region of the longest uORF identified computationally (n = 2,020 with uORF ≥60 nt). The plot is displayed as in Fig. 1G. (B) RF coverage (RF#1) on S1 class bZIP transcription factor genes with multiple uORFs. The scale maximum read value is the same as in Fig. 2C. (C) Tabulation of change in ratio of RFs on the mORF relative to the uORF under normoxia and hypoxia. (D) Comparison of RF (RF#1 and TRAP-RF) and total mRNA-seq reads on SUPPRESSOR OF ACAULIS 51 (SAC51).
Fig. 4.
Fig. 4.
RF profiling reveals conditional alternative splicing. (A) Fold change (FC) in response to hypoxia of RF read number on coding sequences compared with introns. Only introns with ≥10 reads under both treatments are represented. Genes in red had significantly changed RFs on introns (|Fold Change| >2.0, FDR of ≤0.1). Genes discussed in the main text are denoted. (B) RF coverage on selected genes. U1-70K, SMALL NUCLEAR RIBONUCLEOPARTICLE 70K (AT3G50670); GRP7, GLYCINE-RICH RNA-BINDING PROTEIN (AT2G21660). dsRNA read data are from Zheng et al. (46). Gene transcript (blue diagrams) and protein predictions (red diagrams) are shown. The red arrowhead indicates a retained intron.
Fig. 5.
Fig. 5.
RF profiling exposes noncoding mRNAs associated with ribosomes. (A) RF coverage on a lincRNA (At5NC0011780) encoding an uncharacterized structured RNA and a snoRNA, a small ORF RNA (sORF0629/AT1G31935), and a lincRNA with no ORF (AT3G64540). (B) Strand-specific RFs (individual reads) on a NAT encoding a small ORF (seedGroup6045) and a NAT (AT2G05812) overlapping an active protein-coding gene (AT2G05810). Red reads are oriented from left to right (5′ to 3′), and blue reads are oriented from right to left (5′ to 3′). dsRNA read data are from Zheng et al. (46). Blue boxes represent the RNA, and red boxes show the position of ORFs.
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