Article Figures & Data
Figures
- 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 OD254nm. 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 SOVLER 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.
Additional Files
Supplemental Data
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- Supplemental Data - Supplemental Figures 1-5
- Supplemental Data - Supplemental Figures 1-5
