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DHH1/DDX6-like RNA helicases maintain ephemeral half-lives of stress-response mRNAs

Nature Plants volume 6, pages 675–685 (2020)Cite this article

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Abstract

Gene transcription is counterbalanced by messenger RNA decay processes that regulate transcript quality and quantity. We show here that the evolutionarily conserved DHH1/DDX6-like RNA hellicases of Arabidopsis thaliana control the ephemerality of a subset of cellular mRNAs. These RNA helicases co-localize with key markers of processing bodies and stress granules and contribute to their subcellular dynamics. They function to limit the precocious accumulation and ribosome association of stress-responsive mRNAs involved in auto-immunity and growth inhibition under non-stress conditions. Given the conservation of this RNA helicase subfamily, they may control basal levels of conditionally regulated mRNAs in diverse eukaryotes, accelerating responses without penalty.

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Fig. 1: Arabidopsis RH6, RH8 and RH12 overlap in their contribution to growth and development.
Fig. 2: RH6, RH8 and RH12 form cytoplasmic mRNPs contributing to PB and SG formation.
Fig. 3: Attenuation of RH6, RH8 and RH12 function shifts the seedling steady-state transcriptome and translatome from a general growth to stress-responsive state.
Fig. 4: RHs facilitate decay of short-lived mRNAs.
Fig. 5: Stabilization of stress-response mRNAs in the rh6812 genotype is associated with an increase in abundance and translational status.
Fig. 6: The triple rh6812 mutant exhibits a constitutive immune response.

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Data availability

Sequence data are deposited in GEO accession no. GSE136713. All other data needed to evaluate the conclusions are in the Supplementary Information. Source Data for Figs. 1, 2 and 6, and Extended Data Figs. 2–5 and 7 are provided with the paper. Sequence Read Archive, http://www.ncbi.nlm.nih.gov/sra; NCBI Gene Expression Omnibus, https://www.ncbi.nlm.nih.gov/geo/.

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Acknowledgements

We thank J. Parker for kindly providing eds1-2 and pad4-1 seeds, M. Crespi for rdr6sgs2-1 and sgs3-1 and Y. Watanabe for proDCP2:DCP2-GFP in dcp2-1. We thank C. Bousquet-Antonelli for sharing RH6/8/12 antisera. We thank the members of the Bailey-Serres and Sieburth laboratories for discussion. This research was supported by the United States National Science Foundation (grant nos. MCB-1021969 to J.B.-S. and L.E.S. and MCB-1716913 to J.B.-S.) and a UC MacArthur Foundation Chair award to J.B.-S. T.C. was supported by a Royal Thai Government Development and Promotion of Science and Technology Talents Project scholarship.

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Authors and Affiliations

  1. Center for Plant Cell Biology, Department of Botany and Plant Sciences, University of California, Riverside, Riverside, CA, USA

    Thanin Chantarachot, Maureen Hummel, Haiyan Ke, Alek T. Kettenburg, Daniel Chen, Karen Aiyetiwa, Katayoon Dehesh, Thomas Eulgem & Julia Bailey-Serres

  2. School of Biological Sciences, University of Utah, Salt Lake City, UT, USA

    Reed S. Sorenson & Leslie E. Sieburth

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  1. Thanin Chantarachot
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Contributions

T.C., R.S.S., L.S. and J.B.-S. conceived and designed experiments. T.C., R.S., M.H., H.K., A.T.K. and K.A. performed experiments. D.C. contributed to the preparation of genetic material. K.D., T.E., L.S. and J.B.-S. provided reagents. T.C., R.S.S., M.H., L.S. and J.B.-S. analysed data. T.C., R.S.S., L.S. and J.B.-S. wrote the manuscript.

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Correspondence to Julia Bailey-Serres.

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Peer review information Nature Plants thanks Yuichiro Watanabe and the other, anonymous, reviewers for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Phylogenetic relationship and schematic representation of the primary sequence structure of eukaryotic DHH1/DDX6-like DEAD-box RNA helicases.

The evolutionary history was inferred from 58 representative DHH1/DDX6-like helicases across different eukaryotic lineages. The sequences were aligned using the Muscle algorithm, and the phylogenetic tree was generated with the Maximum Likelihood method with 1000 bootstrap replicates. Evolutionary rate differences among sites were modeled with a discrete Gamma distribution. The tree is shown to scale, with branch lengths measured as the number of substitutions per site. Black rectangular boxes on the tree branches depict bootstrap values greater than 50%, with the size of the boxes proportional to the bootstrap values. Grey boxes represent the N- and C-terminal extensions; blue boxes, RecA-like domains; yellow boxes, linker regions.

Extended Data Fig. 2 Characterization of Arabidopsis rh6-1, rh8-1 and rh12-2 mutants and their higher-order mutant combinations.

a, Schematic representation of the RH6, RH8 and RH12 loci and their associated T-DNA insertion alleles. Untranslated and coding regions are depicted as white and black boxes, respectively. Introns are depicted as lines. ‘ATG’ and ‘TAA’ indicate the translational initiation and stop codon, respectively. Inverted white triangles refer to the positions of T-DNA insertions in the rh6-1, rh8-1 and rh12-2 alleles. Arrows indicate the orientation and approximate position of primers used for molecular genotyping of the genes or the T-DNAs: blue, forward primers; red, reverse primers. b, PCR-based genotyping of the homozygous rh6-1, rh8-1 and rh12-2 alleles. Gene and T-DNA-specific primers represented in a were used for PCR amplification of genomic DNA from Col-0 and homozygous rh6-1, rh8-1 and rh12-2 insertion alleles. Results validated with other primers. c, Reverse transcriptase (RT)-PCR analysis of RH6, RH8 and RH12 transcripts. Primers flanking the T-DNAs as indicated in a were used for PCR-based specific detection of RH6, RH8 and RH12 transcripts from the homozygous rh6-1, rh8-1 and rh12-2 alleles in comparison to Col-0. UBQ10 was used as an internal control. d, RT-qPCR analysis of RH6, RH8 and RH12 transcript levels in 7-day-old seedlings of the homozygous rh6-1, rh8-1 and rh12-2 mutants. Gene-specific primers located downstream of the T-DNA as indicated in a were used. Relative transcript fold-change was calculated using PP2AA2 as a reference. Error bars, s.d. (n = 3). Statistical significance was determined by ANOVA, followed by Tukey’s HSD test. Means that are significantly different from each other (P < 0.05) are denoted by different letters. See Source Data for fold change and P values. e, Western blot analysis of RH6, RH8 and RH12 protein levels in 5-day-old seedlings of the homozygous rh6-1, rh8-1 and rh12-2 mutants. f, Western blot analysis of RH6, RH8 and RH12 protein levels in rosette leaves of 32-day-old plants in the double (rh68, rh612 and rh812), double homozygous hemizygous (rh6+/−812, rh68+/−12 and rh6812+/−) and triple (rh6812) mutants. For e,f, each lane was loaded with an equal quantity of protein from a crude homogenate, RH specific antisera were used20. The molecular weight marker (M) ladder and Ponceau S staining as loading control were imaged in visible light. Data are representative of two experiments.

Source data

Extended Data Fig. 3 Reduced expression of RH6, RH8 and RH12 affects plant growth.

a, Schematic diagram and sequences of a 21-nucleotide artificial miRNA and its target site on RH6 expressed in the Arabidopsis MIR319a backbone under the 35S promoter. b, Levels of the artificial amiRH6 in 7-day-old seedlings of three independent amiRH6 lines generated in rh812 background (amiRH6 rh812) determined by pulsed stem-loop RT-qPCR. miRNA fold-change was calculated relative to line #21 using U6 RNA as a reference. Error bars, s.d. (n = 3); n.d., not detectable. c, RT-qPCR analysis of RH6, RH8 and RH12 transcript levels in 7-day-old seedlings of Col-0, rh812 and three amiRH6 rh812 lines. Relative transcript fold-change was calculated using PP2AA2 as a reference. Error bars, s.d. (n = 3). d, Representative rosette growth phenotype of 28-day-old plants of Col-0, rh812 and the amiRH6 rh812 lines. e,, f, Rosette diameters (n = 24) and fresh weights (n = 18-24) of 28-day-old plants of the genotypes presented in d. g, h, Phenotype (g) and primary root lengths (h, n = 27) of 7-day-old seedlings of the wild-type Col-0, the double rh812 mutant and the amiRH6 rh812 lines. Statistical significance was determined by ANOVA, followed by Tukey’s HSD test. Means that are significantly different from each other (P < 0.05) are denoted by different letters. For e, f and h, boxplot boundaries represent the first and third quartiles; a horizontal line divides the interquartile range, median; red diamond, mean. See Source Data for exact sample sizes, fold change or P values for b, c, e, f and h.

Source data

Extended Data Fig. 4 Complementation of the rh6812 phenotype by a genomic fragment of RH6 with a C-terminal FLAG epitope tag (gRH6-FLAG).

a, Rosette growth phenotype of three independent rh6812 gRH6-FLAG lines in the T2 population. 28-day-old plants are shown in comparison to the triple rh6812 mutant and the wild-type Col-0. The rh6812 genotype does not flower (0/36 plants), whereas rh6812 gRH6-FLAG plants are fertile (36/36 plants). Lower panels are magnified images of framed areas in the upper panels. b, Western blot analysis of the RH6-FLAG protein in three independent rh6812 gRH6-FLAG lines presented in a using anti-FLAG M2 antibody. Numbers represent individual plants in the T2 population. c-e, RH6, RH8 and RH12 protein abundance in the rh6812 and rh6812 gRH6-FLAG-22 genotypes. Western blot analysis of RH6 (c, upper panel), RH8 (d, upper panel), RH12 (c, upper panel) and RPS6 (a, b and c, lower panel). Protein levels were determined in triplicate (n = 3) in 5-day-old seedling tissues of Col-0 pro35S:HF-GFP-RPL18 and rh6812 pro35S:HF-GFP-RPL18 and 7-day-old seedling tissues of rh6812 gRH6-FLAG-22 homozygotes. RH specific antisera were used. Data are representative of two experiments.

Source data

Extended Data Fig. 5 The triple rh6812 mutant phenotype is RNA-DEPENDENT RNA POLYMERASE 6 and SUPPRESSOR OF GENE SILENCING 3 independent.

a, Representative rosette growth phenotype of 28-day-old plants of Col-0, single rdr6sgs2-1 and sgs3-1 mutants, triple rh6812 mutant, and quadruple rh6812 rdr6sgs2-1 and rh6812 sgs3-1 mutants. b, Rosette diameter and fresh weight of 28-day-old plants of the genotypes presented in a. Boxplot boundaries represent the first and third quartiles; a horizontal line divides the interquartile range, median; red diamond, mean. Statistical significance was determined by ANOVA (n = 30), followed by Tukey’s HSD test. Data were log-transformed. Means that are significantly different from one another (P < 0.05) are denoted by different letters. Data are representative of two experiments. See Source Data for P values.

Source data

Extended Data Fig. 6 The triple rh6812 mutant transcriptome and translatome are enriched with stress/defense-responsive mRNAs but depleted of those required for growth and development.

a, Volcano plots of change in Total and TRAP mRNA abundance of rh6812 relative to Col-0 based on differential abundance analysis by edgeR. The log2 fold-change (FC) is shown on the x-axis, and the negative log10 of the false-discovery rate (FDR) is shown on the y-axis. Genes with |log2 FC| ≥ 1 and FDR < 0.01 were considered differentially expressed. The total number of genes in the analysis is given in parentheses. b, GO functional categories (biological process) of gene transcripts enriched or depleted in rh6812 relative to Col-0 as identified in a. Twenty non-redundant terms determined by a hypergeometric test with the lowest Bonferroni-adjusted P values presented. Fold enrichment (fold enrich.) represents the number of genes observed relative to the expected number in each category. c, Volcano plots of change in translational status calculated by comparison of steady-state Total and TRAP mRNA abundance in Col-0 and rh6812 based on differential abundance analysis by edgeR. The log2 FC is shown on the x-axis, and the negative log10 of the FDR is shown on the y-axis. Genes with |log2 FC| ≥ 1, FDR < 0.01 were considered enhanced or repressed ribosome-association. The total number of genes used in the analysis is presented in parentheses.

Extended Data Fig. 7 The rh6812 mutant exhibits autoimmunity in a PAD4-partially dependent but EDS1-independent manner.

a, Heatmap representing relative fold change in Total and TRAP mRNA abundance for 104 ‘core’ EDS1/PAD-induced genes33 in rh6812 relative to Col-0. Individual genes are presented as a column with the upper two rows showing their log2 FC following 12 and 24 hours of EDS1 and PAD4 overexpression33, whereas the lower two rows representing their log2 FC in Total and TRAP populations of rh6812 relative to Col-0. b, Representative rosette growth phenotype of 28-day-old plants of Col-0, eds1-2, pad4-1, rh6812, rh6812 eds1-2, rh6812 pad4-1, rh6(+/−)812, rh6(+/−)812 eds1-2 and rh6(+/−)812 pad4-1 genotypes. c, Rosette diameter and fresh weight of 28-day-old plants (n = 12 or 30) of the genotypes presented in b. Boxplot boundaries represent the first and third quartiles; a horizontal line divides the interquartile range, median; red diamond, mean. d, RT-qPCR analysis of PR1 transcripts in 7-day-old seedlings of the rh6812 eds1-2 and rh6812 pad4-1 genotypes relative to rh6812 and the control genotypes Col-0, eds1-2, and pad4-1. Relative transcript fold-change was calculated using ACT1 as a reference. Error bars, s.d. (n = 3). e, Comparison of SA levels in 7-day-old seedlings of the genotypes as in d. Error bars, s.d. (n = 3). Statistical significance in c-e was determined by ANOVA, followed by Tukey’s HSD test. Data in c-e were log-transformed. Significantly differences of means (P < 0.05) are represented by different letters. See Source Data for P values for c, d and e.

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Uncropped immunoblots for Extended Data Fig. 4c–e.

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Chantarachot, T., Sorenson, R.S., Hummel, M. et al. DHH1/DDX6-like RNA helicases maintain ephemeral half-lives of stress-response mRNAs. Nat. Plants 6, 675–685 (2020). https://doi.org/10.1038/s41477-020-0681-8

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