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. 2019 Sep 26;47(17):9216-9230.
doi: 10.1093/nar/gkz712.

RNA degradomes reveal substrates and importance for dark and nitrogen stress responses of Arabidopsis XRN4

Vinay K Nagarajan  1 , Patrick M Kukulich  1 , Bryan von Hagel  1 , Pamela J Green  1
Affiliations

RNA degradomes reveal substrates and importance for dark and nitrogen stress responses of Arabidopsis XRN4

Vinay K Nagarajan et al. Nucleic Acids Res. .

Abstract

XRN4, the plant cytoplasmic homolog of yeast and metazoan XRN1, catalyzes exoribonucleolytic degradation of uncapped mRNAs from the 5' end. Most studies of cytoplasmic XRN substrates have focused on polyadenylated transcripts, although many substrates are likely first deadenylated. Here, we report the global investigation of XRN4 substrates in both polyadenylated and nonpolyadenylated RNA to better understand the impact of the enzyme in Arabidopsis. RNA degradome analysis demonstrated that xrn4 mutants overaccumulate many more decapped deadenylated intermediates than those that are polyadenylated. Among these XRN4 substrates that have 5' ends precisely at cap sites, those associated with photosynthesis, nitrogen responses and auxin responses were enriched. Moreover, xrn4 was found to be defective in the dark stress response and lateral root growth during N resupply, demonstrating that XRN4 is required during both processes. XRN4 also contributes to nonsense-mediated decay (NMD) and xrn4 accumulates 3' fragments of select NMD targets, despite the lack of the metazoan endoribonuclease SMG6 in plants. Beyond demonstrating that XRN4 is a major player in multiple decay pathways, this study identified intriguing molecular impacts of the enzyme, including those that led to new insights about mRNA decay and discovery of functional contributions at the whole-plant level.

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Figures

Figure 1.
Figure 1.
Elevation of many nonpolyadenylated transcripts in xrn4 seedlings. (A) Histograms show the number of protein-coding transcripts that are either increased or decreased (≥2 fold i.e. log2≥ ±1); FDR-adjusted P< 0.05) in two xrn4 mutants relative to the WT (Col-0) in polyA+ and polyA- RNA-seq. Overlap, number of differentially accumulating transcripts that are in common between the mutants. (B) The heat map illustrates the fold changes in the xrn4 mutants of differentially accumulating transcripts (Supplementary Dataset S1). (C) Box plots show the range of fold changes in xrn4 mutants for differentially accumulating transcripts described in panel (B).
Figure 2.
Figure 2.
Most 3′ fragments of cleaved miRNA targets overaccumulate in xrn4 as polyadenylated RNA. (A) Decay plots (D-plots) of a validated miRNA target transcript ARF10 that overaccumulates 3′ fragment of miR160-guided cleavage in xrn4–5. Black arrow, miRNA-guided cleavage site. (B) The heat map of miRNA targets shows fold changes of 3′ fragment abundance in xrn4 in polyA+ and polyA- PARE (Supplementary Table S3). Abundance at cleavage site (±1 nt) in both xrn4 mutants ≥ 10 TP20M and ≥1.5-fold change.
Figure 3.
Figure 3.
XRN4 substrates include decapped intermediates and 3′ fragments of select RNAs. (A) D-plots of IAA2 and SHY2/IAA3 mRNAs show profiles from C-PARE (WT, top) and polyA- PARE (xrn4–5, bottom). Red open circles indicate the cap sites (top) and polyA- MaxSeqs that precisely coincide with cap sites (bottom). (B) Weighted Venn diagrams show overlap between xrn4–5 and xrn4–6, of elevated MaxSeqs that match exactly to cap sites and represent decapped intermediates from unique transcripts in polyA+ or polyA- PARE libraries. The overlap between the two mutants corresponds to decapped XRN4 substrates resulting from the pipeline in Supplementary Figure S7. (C) Overlap between NMD-sensitive transcripts and decapped XRN4 substrates. (D) Overlap between NMD-sensitive transcripts and 3′ fragments overaccumulating in xrn4 polyA+ PARE. For (C and D), CPuORFs, and upf1- and upf3-elevated datasets are described in Supplementary Table S4. Y-axis, z-scores were generated by the GeneSect program and significant overlaps between datasets are indicated by P-values (**, P< 0.01; ***, P < 0.001). For panel (D), in both xrn4 mutants MaxSeq at the same site, abundance ≥ 20 TP20M and ≥2 fold change as described in Supplementary Experimental Procedures.
Figure 4.
Figure 4.
xrn4 mutants show enhanced accumulation of 3′ fragment of an NMD target RNA, eRF1–1. (A) D-plots show prominent 3′ fragment of eRF1–1 RNA in WT and xrn4–5 polyA+ PARE. Blue arrow, MaxSeq in xrn4 and its corresponding position (1511 nt) in WT. Structure of eRF1–1 mRNA: exons in alternating gray and white box; TC1 and TC2, stop codons; yellow, read through element. Black bars, position of 5′ and 3′ probes used in northern blots. (B) RNA levels of full-length (FL) and 3′ fragment (3′) of eRF1–1 in WT, xrn4–5 and xrn4–6 seedlings. (C) Detection of eRF1–1 3′ fragment in WT, and xrn4–5 seedlings by either 5′ (left) or 3′ (right) probe (described in A). (D) Levels of eRF1–1 RNA in WT, upf1–1 and xrn4–5 flowers. RNA levels of NMD marker, SMG7 are also presented (B and D). Northern blots are from total (B and C) or polyA+ (D) RNA samples. Values indicate fold-changes normalized to eIF-4A levels and WT abundance set to 1.
Figure 5.
Figure 5.
Decapped XRN4 substrates are over-represented for categories associated with photosynthesis, stress and hormone responses. (A) Transcripts overaccumulating decapped intermediates in both xrn4–5 and xrn4–6 were identified from the overlap data in Figure 3B. Weighted Venn diagrams designate these as decapped polyA+ (polyadenylated) and polyA- (deadenylated) XRN4 substrates as discussed in the text. (B) Gene-ontology (GO) enrichment categories of decapped XRN4 substrates. Highlighted are significant GO functions associated with photosynthesis (green), abiotic stimulus/responses (orange), hormone responses (blue) and other categories (gray). Only 15 most significant terms are presented for polyA- and the rest are listed in Supplementary Table S5 (PARE). (C) Nitrogen (N) responsive and NEC transcripts are over-represented among XRN4 substrates. Overlap significance between decapped XRN4 substrates (**, P < 0.01; ***, P < 0.001) and transcripts from different datasets was evaluated using the GeneSect program (Supplementary Table S4).
Figure 6.
Figure 6.
xrn4 is oversensitive to dark treatment of leaves and seedlings. Detached leaves (A) and intact seedlings (B) of WT, xrn4–5 and complemented line of xrn4–5 (Comp) after extended dark (A, 4 d and B, 12 d) treatments; scale bar, 20 mm. (C) Chlorophyll content of dark treated (leaves, 4 d and seedlings, 12 d) WT, xrn4–5, xrn4–6 and Comp presented as a percentage of diurnal (100%). Histograms are means from three biological replicates; four leaves from a total of 12 plants per genotype per condition (A) and a total of 15 pools of seedlings (5 to 6 plants per pool) per genotype per condition (B) were assayed and presented as percentages ± standard error of the mean (SEM). (D) Quantitative RT-PCR of polyA+ RNA show mRNA levels of ATL8, bZIP1 and eIF-4A (control transcript) in 2-week-old seedlings of WT and xrn4–5 after 0 h and 24 h of dark treatment. WT levels at 0 h set to 1. Data are means ± standard deviation (SD) from four biological replicates; **, P< 0.01 and *, P< 0.05.
Figure 7.
Figure 7.
xrn4 mutants show attenuated lateral root growth after recovery from N starvation. (A) Root images showing LR phenotype of WT, xrn4–5 and its complemented line after 7 d of recovery from 7 d of N starvation. (B) Number of LR for WT and xrn4 mutants from three independent experiments are shown (N = 12, 26 and 15 seedlings per genotype). Data are means ± SEM. (C) RNA half-life (t1/2) of IAA2 and SHY2/IAA3 under control conditions. Total RNA northern blots show time course of RNA abundance in WT and xrn4–5 after cordycepin treatment. Average t1/2 in min ± SD of two biological replicates is below. RNA levels of stable transcript eIF-4A are also shown. (D) Quantitative splinted-ligation RT-PCR of total RNA shows levels of decapped (5′-P) and capped (5′ Cap, CIP + TAP) RNAs of IAA2 and SHY2/IAA3 in WT and xrn4–5 seedlings. Levels of control RNAs are in Supplementary Figure S13. Histograms are means ± SD of four biological replicates; **, P < 0.01 and *, P < 0.05.
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