Monitoring of XRN4 Targets Reveals the Importance of Cotranslational Decay during Arabidopsis Development

Abstract

RNA turnover is a general process that maintains appropriate mRNA abundance at the posttranscriptional level. Although long thought to be antagonistic to translation, discovery of the 5' to 3' cotranslational mRNA decay pathway demonstrated that both processes are intertwined. Cotranslational mRNA decay globally shapes the transcriptome in different organisms and in response to stress; however, the dynamics of this process during plant development is poorly understood. In this study, we used a multiomics approach to reveal the global landscape of cotranslational mRNA decay during Arabidopsis (Arabidopsis thaliana) seedling development. We demonstrated that cotranslational mRNA decay is regulated by developmental cues. Using the EXORIBONUCLEASE4 (XRN4) loss-of-function mutant, we showed that XRN4 poly(A⁺) mRNA targets are largely subject to cotranslational decay during plant development. As cotranslational mRNA decay is interconnected with translation, we also assessed its role in translation efficiency. We discovered that clusters of transcripts were specifically subjected to cotranslational decay in a developmental-dependent manner to modulate their translation efficiency. Our approach allowed the determination of a cotranslational decay efficiency that could be an alternative to other methods to assess transcript translation efficiency. Thus, our results demonstrate the prevalence of cotranslational mRNA decay in plant development and its role in translational control.

Figure 1.

XRN4 differentially accumulates in polysomes during seedling development. A, Polysomal extracts prepared from 3-, 7-, 15-, and 25-d-old seedlings were fractionated on a Suc gradient, and polysome traces were obtained through measurement of OD_254nm. Polysome profiling was performed starting from identical quantities of N₂-pulverized tissues (e.g. 300 mg of biomass). B, Total proteins extracted from polysomal and input fractions were analyzed by immunoblotting. The four blots were probed with an antibody specific to XRN4. Inputs correspond to an equivalent of 10 mg of tissue powder for all stages. For polysomal fractions, loaded proteins were precipitated from identical volumes of each fraction. Data are representative of at least three replicates.

Figure 2.

XRN4 loss-of-function mutation has minimal impact during seedling development on total and polysome RNA levels. FCs between xrn4-5 and the Col-0 wild type (wt) were calculated for total (A) and polysome (B) RNA in each condition. The log₂ value of the mean is represented in each graph. The number of transcripts significantly regulated in xrn4-5 is reported (as differentially expressed genes [DEG] in gray for upregulated and downregulated transcripts) and was calculated using DESeq2. Dashed red lines mark the |log₂(2)| values.

Figure 3.

Metagene analyses displaying the abundance of 5ʹP reads relative to stop codons. B, Wild-type (wt) 3-d-old stage. C, Wild-type 7-d-old stage. D, Wild-type 15-d-old stage. E, Wild-type 25-d-old stage. F, xrn4-5 3-d-old stage. G, xrn4-5 7-d-old stage. H, xrn4-5 15-d-old stage. I, xrn4-5 25-d-old stage. Data are means ± sd. The illustrations (A) represent 5ʹP intermediate accumulation at –47 and –17 nucleotides (nt) before stop codons. RPM, Reads per million.

Figure 4.

Identification and features of XRN4 cotranslational decay targets during development. A to D, Volcano plots of the change in read abundance in xrn4-5 over wild-type Col-0 (wt). Vertical red dashed lines mark the |log₂(2)| values. Log₂ FC and Benjamini-Hochberg adjusted P values (BH) were calculated through the DESeq2 pipeline (as DEGs in blue for up-regulated and in red for downregulated transcripts). Horizontal solid black lines demarcate adjusted P values of 0.05. E, Venn diagram of cotranslational decay targets during development. F and G, The majority of XRN4 cotranslational decay targets show longer RNA half-lives in vcs-7 and vcs-7 SOV^LER mutants (F) and the rh6812 mutant (G). RNA half-lives were collected from Sorenson et al. (2018; F) or from Chantarachot et al. (2020; G). Only transcripts present in each data set are represented (n = 444/565 for F and n = 390/565 for G). H to J, Intron number (H), CDS length (I), and proportion of AU motifs (J) in the 5ʹ-UTR of transcripts targeted by XRN4 compared with nontargeted random transcripts (n = 565). Asterisks indicate significant difference (***P < 0.001 and *P < 0.05). n.s., Nonsignificant.

Figure 5.

Cotranslational decay is regulated during development and influences protein production. A, Transcript variation at the polysome level during development using 3-d samples as a reference (n = 3,366). B, Transcript variation at the degradome level during development using 3-d samples as a reference (n = 3,366). Gray lines represent individual transcript variation. Transcript distribution is represented by notched box plots, and significance was assessed by P values (nonparametric Wilcoxon test). C, Heat map of cotranslational decay efficiency (ratio in degradome data over polysome RNA-seq data; n = 3,366). Red values correspond to high decay efficiency and yellow values to low decay efficiency. D and E, Immunoblots using LUT1 (D) and CDC2 (E) antibodies. Both candidates were analyzed on distinct SDS-PAGE gels (8% and 10% acrylamide, respectively). RPL13 and UGPase antibodies were used as loading controls. Each immunoblot was performed as two biological replicates.