Impact of poly(A)-tail G-content on Arabidopsis PAB binding and their role in enhancing translational efficiency

Abstract

Background: Polyadenylation plays a key role in producing mature mRNAs in eukaryotes. It is widely believed that the poly(A)-binding proteins (PABs) uniformly bind to poly(A)-tailed mRNAs, regulating their stability and translational efficiency.

Results: We observe that the homozygous triple mutant of broadly expressed Arabidopsis thaliana PABs, AtPAB2, AtPAB4, and AtPAB8, is embryonic lethal. To understand the molecular basis, we characterize the RNA-binding landscape of these PABs. The AtPAB-binding efficiency varies over one order of magnitude among genes. To identify the sequences accounting for the variation, we perform poly(A)-seq that directly sequences the full-length poly(A) tails. More than 10% of poly(A) tails contain at least one guanosine (G); among them, the G-content varies from 0.8 to 28%. These guanosines frequently divide poly(A) tails into interspersed A-tracts and therefore cause the variation in the AtPAB-binding efficiency among genes. Ribo-seq and genome-wide RNA stability assays show that AtPAB-binding efficiency of a gene is positively correlated with translational efficiency rather than mRNA stability. Consistently, genes with stronger AtPAB binding exhibit a greater reduction in translational efficiency when AtPAB is depleted.

Conclusions: Our study provides a new mechanism that translational efficiency of a gene can be regulated through the G-content-dependent PAB binding, paving the way for a better understanding of poly(A) tail-associated regulation of gene expression.

Keywords: Arabidopsis; PAB binding efficiency; Poly(A) tails; Poly(A)-binding proteins; Poly(A)-tail G-content; Translational efficiency; mRNA stability.

Fig. 1

AtPABs play essential roles in plants. a Heterozygous atpab triple mutants exhibited various phenotypic abnormalities, such as dwarf, multi-branches, premature senility, serrated leaves, and sterility. b About 25% aborted ovules (indicated by red triangles) were observed in the siliques of the mut (atpab2^+/− atpab4 atpab8). c Embryos of atpab2 atpab4 atpab8 degenerated when normal embryos reached the cotyledon stage. Embryos were obtained from a single silique of the mut

Fig. 2

mRNAs bind to AtPABs with different efficiencies. a Schematic of the CLIP-seq experiment, which detects the direct RNA-binding targets of AtPABs. b AtPABs bind predominantly to consecutive A’s. Reads with ≥ 12 consecutive N’s were counted because the yeast Pab1p requires at least 12 consecutive A’s for binding. c mRNA is the major binding target of AtPABs. d CLIP-seq reads were mainly mapped to the 3′-ends of mRNAs. The length of each mRNA was scaled to 100%. “0” and “100” represent the transcription start and end, respectively. e The AtPAB-binding gene detected in CLIP-seq was validated by RIP-RT-PCR. The distribution of the AtPAB-CLIP reads is shown by the wiggle plots. The gene model shows the untranslated regions (gray boxes), coding sequences (black boxes), and introns (lines). The input and IP panels show the mRNA level of an AtPAB-binding gene in total RNA and RIP experiments (anti-HA antibody), respectively. The minus sign (−) indicates the negative control (the wild-type Col) in which the GFP-HA tagged AtPAB is absent. f The AtPAB2-binding efficiency significantly varies among genes in Col. Each dot represents a target gene of AtPAB2. The diagonal is shown by a blue dashed line. g The difference in AtPAB2-binding efficiency among genes was validated by RIP-RT-qPCR. Green bars and purple bars show the AtPAB2-binding efficiency estimated by CLIP-seq and RIP-RT-qPCR, respectively. The ~ 10-fold difference in AtPAB2-binding efficiency between At1G12110 and At4G40090 that was detected by CLIP-seq was validated by RIP-RT-qPCR. The error bar represents the standard deviation of three replicates. h The binding efficiencies of genes were highly correlated among AtPABs. P values were given by Pearson’s correlation analysis (P < 1 × 10⁻¹⁰⁰ in all three pairwise comparisons)

Fig. 3

G-content contributes to the binding variance of AtPABs. a Schematic of the poly(A)-seq experiment that directly sequences the poly(A) tails of mRNAs. High-quality reads with recognizable Illumina 3′-adaptor sequences were used for further analysis. b Example reads from the poly(A)-seq show different propensities for AtPAB binding among genes. Yeast Pab1p and human PABP require at least 12 and 11 consecutive A’s for binding, respectively. Guanosines can inhibit AtPAB binding by cutting the poly(A) tail into fragments with < 12 consecutive A’s. Ref represents the reference Arabidopsis genome. c The G% was negatively correlated with the AtPAB-binding efficiency of a gene. Genes were divided into ten equal-size groups according to the average G% in the poly(A) tails. P values were given by the Spearman’s correlation. d Paralogous genes with lower G% in the poly(A) tail exhibited higher AtPAB2-binding efficiencies than their within-species paralogs with higher G% in the poly(A) tails. P values were given by the paired Mann-Whitney U test

Fig. 4

AtPAB-binding efficiency positively correlates with translational efficiency. a The possible consequences of AtPAB binding to mRNAs. b The AtPAB-binding efficiency was poorly correlated with the mRNA degradation rate in Col. c The AtPAB-binding efficiency was positively correlated with the translational efficiency (TE) in Col

Fig. 5

AtPABs enhance translational efficiency. a Polysome profiling shows the global reduction in TE in the mut. The x-axis indicates the detecting distance from 0 to 75 mm of the 5–50% sucrose gradient. b, c Genes showing reduced TE in the mut (b) exhibited significantly higher AtPAB2-binding efficiencies (c). The TE was calculated with the ribo-seq data. Genes with decreased TE in the mut (brown dots) were defined as those with TE fold change smaller than the median. P value in c was given by the Mann-Whitney U test. d Genes with exclusive adenosines in the poly(A) tail tended to be present in the gene group showing the downregulation of TE in the mut (defined in b). e Schematic of the TMT (Tandem Mass Tag)-based quantitative proteomics analysis. Protein samples extracted from three biological replicates of Col and the mut were labeled with TMT 6 plex reagent separately. f–h The protein synthesis rate was calculated as the protein level of a gene normalized to its mRNA level, and similar results were obtained as those in b–d