Regulation of co-translational mRNA decay by PAP and DXO1 in Arabidopsis

Abstract

Background: mRNA decay is central in the regulation of mRNA homeostasis in the cell. The recent discovery of a co-translational mRNA decay pathway (also called CTRD) has changed our understanding of the mRNA decay process. This pathway has emerged as an evolutionarily conversed mechanism essential for specific physiological processes in eukaryotes, especially in plants. In Arabidopsis, this pathway is targeted mainly by the exoribonuclease XRN4. However, the details of the molecular regulation of this pathway are still unclear.

Results: In this study, we first tested the role of the 3'-phosphoadenosine 5'-phosphate (PAP), an inhibitor of exoribonucleases in the regulation of CTRD. Using 5'Pseq approach, we discovered that FRY1 inactivation impaired XRN4-CTRD activity. Based on this finding, we demonstrated that exogenous PAP treatment stabilizes CTRD mRNA targets. Furthermore, we also tested the implication of the exoribonuclease DXO1 in CTRD regulation. We found that DXO1, another exoribonuclease sensitive to PAP, is also involved in the CTRD pathway, probably by targeting NAD⁺-capped mRNAs. DXO1 specifically targets mRNAs linked to stress response.

Conclusions: Our study provides further insights into the regulation of CTRD in Arabidopsis and demonstrates that other exoribonucleases can be implicated in this pathway.

Keywords: 3ʹ-phosphoadenosine 5ʹ-phosphate; Arabidopsis; Co-translational mRNA decay; DXO1; FRY1; XRN4.

Fig. 1

XRN4 accumulation to polysomes is impaired in fry1mutant. Polysomal extracts prepared from Col0 or fry1 shoot (A) and root (B) were fractionated on a sucrose gradient. Polysome profiles were obtained by continuous 254 nm absorption measurement (A₂₅₄ expressed in arbitrary units, A.U.). All the profiles were analysed simultaneously. Each fraction was labelled from F1 to F9. C-D. Total proteins extracted from polysomal (F5 to F9) and input fractions were analysed by immunoblotting using XRN4 or RPL13 specific antibodies (C. Shoot samples, D. Root samples). The four blots were prepared and analysed simultaneously. The same quantities of tissues were used for each condition (e.g. 300 mg of biomass)

Fig. 2

FRY1 inactivation affects co-translational mRNA decay in both shoot and root. (A) Metagene analysis of 5′P reads accumulation around stop codons. The different profiles are representative of 3 biological replicates. (B) Distribution of Terminational Stalling Index (TSI) in Col0, xrn4 and fry1 and comparison of XRN4 and FRY1 targets in shoot. (C) Distribution of Terminational Stalling Index (TSI) in Col0, xrn4 and fry1 and comparison of XRN4 and FRY1 targets in roots. Only transcripts with a TSI > 3 in Col0 were kept. A transcript was considered as a XRN4 or FRY1 target when the TSI value in xrn4 and/or fry1 is at least two times lower than in Col0. N = 3 biological replicates. Significance of the distribution was tested using a Wilcoxon test. ***: p-value < 0.001

Fig. 3

PAP treatment affects XRN4 accumulation to polysomes and mRNA stability. A. Total proteins extracted from polysomal and input fractions prepared from (A) Col0 shoot or (B) Col0 root incubated for 1 h on liquid MS medium (untreated) or 1 h on liquid MS medium supplemented with 1 mM PAP. The four blots were prepared and analysed simultaneously. The same quantities of tissues were used for each condition (e.g. 300 mg of biomass). C, D. mRNA stability was determined in vivo after 1 mM cordycepin treatment (blue line) or after 1 mM cordycepin and 1 mM PAP treatment (orange line) followed by RT-ddPCR. Half-lives are indicated on each graph. Mean ± SD. N = 3 biological replicates

Fig. 4

DXO1 catalytic inactivation affects co-translational mRNA decay in both shoot and root. (A) Metagene analysis of 5′P reads accumulation around stop codons. The different profiles are representative of 3 biological replicates. (B) Distribution of Terminational Stalling Index (TSI) in Col0, DXO1(E394A/D396A) and xrn4 and comparison of XRN4 and DXO1 targets in shoot. (C) Distribution of Terminational Stalling Index (TSI) in in Col0, DXO1(E394A/D396A) and xrn4 and comparison of XRN4 and DXO1 targets in root. Only transcripts with a TSI > 3 in Col0 were kept. A transcript was considered as a XRN4 or DXO1 target when the TSI value in xrn4 and/or DXO1(E394A/D396A) is at least two times lower than in Col0. N = 3 biological replicates. Significance of the distribution was tested using a Wilcoxon test. ***: p-value < 0.001

Fig. 5

DXO1 co-translational mRNA decay targets are identified as NAD⁺-capped RNAs and targeted by FRY1. (A) Distribution of Terminational Stalling Index (TSI) of NAD⁺-capped RNAs (identified in Hu et al., 2021) in Col0 and DXO1(E394A/D396A) line, N = 3,011. Transcripts with a TSI value higher than 0 in Col0 were kept for the analysis. Significance of the distribution was tested using a Wilcoxon test. ***: p-value < 0.001. (B) Venn diagram representation of NAD⁺-capped transcripts targeted by CTRD (TSI > 3 in Col0) and transcripts with a lower TSI value in DXO1 (E394A/D396A) compared to Col0. To test the significance of the overlap, hypergeometric test was performed (***: p-value < 0.001). (C) List of response to stimulus GO terms for the overlap targets (N = 1,967). The number of genes per category is indicated. D, E. Distribution of the TSI for the common targets of DXO1(E394A/D396A) and FRY1 in shoot and root respectively. Significance of the distribution was tested using a Wilcoxon test. ***: p-value < 0.001