Post-transcriptional gene silencing (PTGS) is a major mechanism regulating gene expression in higher eukaryotes. To identify novel players in PTGS, a forward genetics screen was performed on an Arabidopsis thaliana line overexpressing a strong growth-repressive gene, ETHYLENE RESPONSE FACTOR6 (ERF6). We identified six independent ethyl-methanesulfonate mutants rescuing the dwarfism of ERF6-overexpressing plants as a result of transgene silencing. Among the causative genes, ETHYLENE-INSENSITIVE5, SUPERKILLER2 and HASTY1 have previously been reported to inhibit PTGS. Notably, the three other causative genes have not, to date, been related to PTGS: UTP:RNA-URIDYLYLTRANSFERASE1 (URT1), C-TERMINAL DOMAIN PHOSPHATASE-LIKE3 (CPL3) and RESURRECTION1 (RST1). We show that these genes may participate in protecting the 3’ end of transgene transcripts. We present a model in which URT1, CPL3 and RST1 are classified as PTGS suppressors, as compromisation of these genes provokes the accumulation of aberrant transcripts which, in turn, trigger the production of small interfering RNAs, initiating RNA silencing.
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The data that support the findings of this study are available from the corresponding author upon request.
Baulcombe, D. RNA silencing in plants. Nature 431, 356–363 (2004).
Chen, X. A silencing safeguard: links between RNA silencing and mRNA processing in Arabidopsis. Dev. Cell 14, 811–812 (2008).
Liu, L. & Chen, X. RNA quality control as a key to suppressing RNA silencing of endogenous genes in plants. Mol. Plant 9, 826–836 (2016).
Hentze, M. W. & Kulozik, A. E. A perfect message: RNA surveillance and nonsense-mediated decay. Cell 96, 307–310 (1999).
Belostotsky, D. mRNA turnover meets RNA interference. Mol. Cell 16, 498–500 (2004).
Parker, R. & Song, H. The enzymes and control of eukaryotic mRNA turnover. Nat. Struct. Mol. Biol. 11, 121–127 (2004).
Dunckley, T. & Parker, R. The DCP2 protein is required for mRNA decapping in Saccharomyces cerevisiae and contains a functional MutT motif. EMBO J. 18, 5411–5422 (1999).
Xu, J., Yang, J.-Y., Niu, Q.-W. & Chua, N.-H. Arabidopsis DCP2, DCP1, and VARICOSE form a decapping complex required for postembryonic development. Plant Cell 18, 3386–3398 (2006).
Zakrzewska-Placzek, M., Souret, F. F., Sobczyk, G. J., Green, P. J. & Kufel, J. Arabidopsis thaliana XRN2 is required for primary cleavage in the pre-ribosomal RNA. Nucleic Acids Res. 38, 4487–4502 (2010).
Souret, F. F., Kastenmayer, J. P. & Green, P. J. AtXRN4 degrades mRNA in Arabidopsis and its substrates include selected miRNA targets. Mol. Cell 15, 173–183 (2004).
Rymarquis, L. A., Souret, F. F. & Green, P. J. Evidence that XRN4, an Arabidopsis homolog of exoribonuclease XRN1, preferentially impacts transcripts with certain sequences or in particular functional categories. RNA 17, 501–511 (2011).
Dorcey, E. et al. Context-dependent dual role of SKI8 homologs in mRNA synthesis and turnover. PLoS Genet. 8, e1002652 (2012).
Anderson, J. S. J. & Parker, R. The 3′ to 5′ degradation of yeast mRNAs is a general mechanism for mRNA turnover that requires the SKI2 DEVH box protein and 3′ to 5′ exonucleases of the exosome complex. EMBO J. 17, 1497–1506 (1998).
Brown, J. T., Bai, X. & Johnson, A. W. The yeast antiviral proteins Ski2p, Ski3p, and Ski8p exist as a complex in vivo. RNA 6, 449–457 (2000).
Hooker, T. S., Lam, P., Zheng, H. & Kunst, L. A core subunit of the RNA-processing/degrading exosome specifically influences cuticular wax biosynthesis in Arabidopsis. Plant Cell 19, 904–913 (2007).
Zhang, X. et al. Suppression of endogenous gene silencing by bidirectional cytoplasmic RNA decay in Arabidopsis. Science 348, 120–123 (2015).
Gazzani, S., Lawrenson, T., Woodward, C., Headon, D. & Sablowski, R. A link between mRNA turnover and RNA interference in Arabidopsis. Science 306, 1046–1048 (2004).
Moreno, A. B. et al. Cytoplasmic and nuclear quality control and turnover of single-stranded RNA modulate post-transcriptional gene silencing in plants. Nucleic Acids Res. 41, 4699–4708 (2013).
Kumakura, N. et al. SGS3 and RDR6 interact and colocalize in cytoplasmic SGS3/RDR6‐bodies. FEBS Lett. 583, 1261–1266 (2009).
Peragine, A., Yoshikawa, M., Wu, G., Albrecht, H. L. & Poethig, R. S. SGS3 and SGS2/SDE1/RDR6 are required for juvenile development and the production of trans-acting siRNAs in Arabidopsis. Genes Dev. 18, 2368–2379 (2004).
Dong, Z., Han, M.-H. & Fedoroff, N. The RNA-binding proteins HYL1 and SE promote accurate in vitro processing of pri-miRNA by DCL1. Proc. Natl Acad. Sci. USA 105, 9970–9975 (2008).
Ketting, R. F. et al. Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans. Genes Dev. 15, 2654–2659 (2001).
Bouché, N., Lauressergues, D., Gasciolli, V. & Vaucheret, H. An antagonistic function for Arabidopsis DCL2 in development and a new function for DCL4 in generating viral siRNAs. EMBO J. 25, 3347–3356 (2006).
Martínez de Alba, A. E. et al. In plants, decapping prevents RDR6-dependent production of small interfering RNAs from endogenous mRNAs. Nucleic Acids Res. 43, 2902–2913 (2015).
Knop, K. et al. Active 5′ splice sites regulate the biogenesis efficiency of Arabidopsis microRNAs derived from intron-containing genes. Nucleic Acids Res. 45, 2757–2775 (2017).
Stepien, A. et al. Posttranscriptional coordination of splicing and miRNA biogenesis in plants. RNA 8, e1403 (2017).
Bartel, D. P. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281–297 (2004).
Bologna, N. G. et al. Nucleo-cytosolic shuttling of ARGONAUTE1 prompts a revised model of the plantmicroRNA pathway. Mol. Cell 69, 709–719 (2018).
Bollman, K. M. et al. HASTY, the Arabidopsis ortholog of exportin 5/MSN5, regulates phase change and morphogenesis. Development 130, 1493–1504 (2003).
Li, S. et al. MicroRNAs inhibit the translation of target mRNAs on the endoplasmic reticulum in Arabidopsis. Cell 153, 562–574 (2013).
Liu, Q., Wang, F. & Axtell, M. J. Analysis of complementarity requirements for plant microRNA targeting using a Nicotiana benthamiana quantitative transient assay. Plant Cell 26, 741–753 (2014).
Carbonell, A. et al. Functional analysis of three Arabidopsis ARGONAUTES using slicer-defective mutants. Plant Cell 24, 3613–3629 (2012).
Carmell, M. A., Xuan, Z., Zhang, M. Q. & Hannon, G. J. The Argonaute family: tentacles that reach into RNAi, developmental control, stem cell maintenance, and tumorigenesis. Genes Dev. 16, 2733–2742 (2002).
Tang, G. siRNA and miRNA: an insight into RISCs. Trends Biochem. Sci. 30, 106–114 (2005).
Vaucheret, H., Mallory, A. C. & Bartel, D. P. AGO1 homeostasis entails coexpression of MIR168 and AGO1 and preferential stabilization of miR168 by AGO1. Mol. Cell 22, 129–136 (2006).
Martínez de Alba, A. E., Jauvion, V., Mallory, A. C., Bouteiller, N. & Vaucheret, H. The miRNA pathway limits AGO1 availability during siRNA-mediated PTGS defense against exogenous RNA. Nucleic Acids Res. 39, 9339–9344 (2011).
Dubois, M. et al. ETHYLENE RESPONSE FACTOR6 acts as a central regulator of leaf growth under water-limiting conditions in Arabidopsis. Plant Physiol. 162, 319–332 (2013).
Mlotshwa, S. et al. Transcriptional silencing induced by Arabidopsis T‐DNA mutants is associated with 35S promoter siRNAs and requires genes involved in siRNA‐mediated chromatin silencing. Plant J. 64, 699–704 (2010).
Elmayan, T. & Vaucheret, H. Expression of single copies of a strongly expressed 35S transgene can be silenced post‐transcriptionally. Plant J. 9, 787–797 (1996).
Elmayan, T. et al. Arabidopsis mutants impaired in cosuppression. Plant Cell 10, 1747–1757 (1998).
Kurihara, Y., Takashi, Y. & Watanabe, Y. The interaction between DCL1 and HYL1 is important for efficient and precise processing of pri-miRNA in plant microRNA biogenesis. RNA 12, 206–212 (2006).
Sement, F. M. et al. Uridylation prevents 3′ trimming of oligoadenylated mRNAs. Nucleic Acids Res. 41, 7115–7127 (2013).
De Almeida, C., Scheer, H., Zuber, H. & Gagliardi, D. RNA uridylation: a key posttranscriptional modification shaping the coding and noncoding transcriptome. RNA 9, e1440 (2018).
Lim, J. et al. Uridylation by TUT4 and TUT7 marks mRNA for degradation. Cell 159, 1365–1376 (2014).
Zuber, H., Scheer, H., Joly, A.-C. & Gagliardi, D. Respective contributions of URT1 and HESO1 to the uridylation of 5′ fragments produced from RISC-cleaved mRNAs. Front. Plant Sci. 9, 1438 (2018).
Li, F. et al. Modulation of RNA polymerase II phosphorylation downstream of pathogen perception orchestrates plant immunity. Cell Host Microbe 16, 748–758 (2014).
Cui, P. et al. The RNA polymerase II C-terminal domain phosphatase-like protein FIERY2/CPL1 interacts with eIF4AIII and is essential for nonsense-mediated mRNA decay in Arabidopsis. Plant Cell 28, 770–785 (2016).
Chen, T. et al. A KH-domain RNA-binding protein interacts with FIERY2/CTD phosphatase-like 1 and splicing factors and is important for pre-mRNA splicing in Arabidopsis. PLoS Genet. 9, e1003875 (2013).
Manavella, P. A. et al. Fast-forward genetics identifies plant CPL phosphatases as regulators of miRNA processing factor HYL1. Cell 151, 859–870 (2012).
Elliott, B. J., Dattaroy, T., Meeks-Midkiff, L. R., Forbes, K. P. & Hunt, A. G. An interaction between an Arabidopsis poly (A) polymerase and a homologue of the 100 kDa subunit of CPSF. Plant Mol. Biol. 51, 373–384 (2003).
Meeks, L. R., Addepalli, B. & Hunt, A. G. Characterization of genes encoding poly (A) polymerases in plants: evidence for duplication and functional specialization. PLoS ONE 4, e8082 (2009).
Davidson, L., Muniz, L. & West, S. 3′ end formation of pre-mRNA and phosphorylation of Ser2 on the RNA polymerase II CTD are reciprocally coupled in human cells. Genes Dev. 28, 342–356 (2014).
Koiwa, H. et al. C-terminal domain phosphatase-like family members (AtCPLs) differentially regulate Arabidopsis thaliana abiotic stress signaling, growth, and development. Proc. Natl Acad. Sci. USA 99, 10893–10898 (2002).
Kastenmayer, J. P. & Green, P. J. Novel features of the XRN-family in Arabidopsis: evidence that AtXRN4, one of several orthologs of nuclear Xrn2p/Rat1p, functions in the cytoplasm. Proc. Natl Acad. Sci. USA 97, 13985–13990 (2000).
Lam, P. et al. RDR1 and SGS3, components of RNA-mediated gene silencing, are required for the regulation of cuticular wax biosynthesis in developing inflorescence stems of Arabidopsis. Plant Physiol. 159, 1385–1395 (2012).
Chen, X. et al. Mutation of the RESURRECTION1 locus of Arabidopsis reveals an association of cuticular wax with embryo development. Plant Physiol. 139, 909–919 (2005).
Mang, H. G. et al. The Arabidopsis RESURRECTION1 gene regulates a novel antagonistic interaction in plant defense to biotrophs and necrotrophs. Plant Physiol. 151, 290–305 (2009).
Lange, H. et al. The RNA helicases AtMTR4 and HEN2 target specific subsets of nuclear transcripts for degradation by the nuclear exosome in Arabidopsis thaliana. PLoS Genet. 10, e1004564 (2014).
Roman, G., Lubarsky, B., Kieber, J. J., Rothenberg, M. & Ecker, J. R. Genetic analysis of ethylene signal transduction in Arabidopsis thaliana: five novel mutant loci integrated into a stress response pathway. Genetics 139, 1393–1409 (1995).
Telfer, A. & Poethig, R. S. HASTY: a gene that regulates the timing of shoot maturation in Arabidopsis thaliana. Development 125, 1889–1898 (1998).
Branscheid, A. et al. SKI2 mediates degradation of RISC 5′-cleavage fragments and prevents secondary siRNA production from miRNA targets in Arabidopsis. Nucleic Acids Res. 43, 10975–10988 (2015).
McElver, J. et al. Insertional mutagenesis of genes required for seed development in Arabidopsis thaliana. Genetics 159, 1751–1763 (2001).
Kilian, J. et al. The AtGenExpress global stress expression data set: protocols, evaluation and model data analysis of UV-B light, drought and cold stress responses. Plant J. 50, 347–363 (2007).
Schneeberger, K. et al. SHOREmap: simultaneous mapping and mutation identification by deep sequencing. Nat. Methods 6, 550–551 (2009).
Dubois, M. et al. The ETHYLENE RESPONSE FACTORS ERF6 and ERF11 antagonistically regulate mannitol-induced growth inhibition in Arabidopsis. Plant Physiol. 169, 166–179 (2015).
Joo, S., Liu, Y., Lueth, A. & Zhang, S. MAPK phosphorylation-induced stabilization of ACS6 protein is mediated by the non-catalytic C-terminal domain, which also contains the cis‐determinant for rapid degradation by the 26S proteasome pathway. Plant J. 54, 129–140 (2008).
Löw, R. in The Nucleic Acid Protocols Handbook (ed. Rapley, R.) 239–247 (Humana Press, 2000).
Derrien, B. et al. A suppressor screen for AGO1 degradation by the viral F-Box P0 protein uncovers a role for AGO DUF1785 in sRNA duplex unwinding. Plant Cell 30, 1353–1374 (2018).
Sement, F. M. & Gagliardi, D. Detection of uridylated mRNAs. Methods Mol. Biol. 1125, 43–51 (2014).
Gallagher, S. R. in GUS Protocols: Using the Gus Gene as a Reporter of Gene Expression (ed. Gallagher, S. R.) 47–59 (Academic Press, 1992).
Dubois, M., Claeys, H., Van den Broeck, L. & Inzé, D. Time of day determines Arabidopsis transcriptome and growth dynamics under mild drought. Plant Cell Environ. 40, 180–189 (2017).
Wang, K., Wang, X., Li, M., Shi, T. & Yang, P. Low genetic diversity and functional constraint of miRNA genes participating pollen–pistil interaction in rice. Plant Mol. Biol. 95, 89–98 (2017).
We thank the Systems Biology of Yield group for providing a stimulating scientific environment; H. Vaucheret, M. Matsui, H. Vanhaeren and L. Van den Broeck for fruitful discussions and/or sharing of plant material; L. De Milde for practical help; and A. Bleys for critically reading and improving the manuscript. This work was supported by Ghent University (Bijzonder Onderzoeksfonds Methusalem, Project No. BOF08/01M00408). M.D. is a post-doctoral fellow of Flanders Research Foundation (No. 12Q7919N).
The authors declare no competing interests.
Journal peer review information: Nature Plants thanks Blake Meyers, Xuehua Zhong and other anonymous reviewer(s) for their contribution to the peer review of this work.
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Supplementary Figures 1–10 and Supplementary Tables 1 and 4–6.
Quantification of rqc-siRNAs reads (CPM) arising from the protein-coding genes in ERF6-GR, urt1sgi2 (a), cpl3sgi3 (b), and rst1sgi6 (c). Three biological repeats for each genotype were performed. The DEseq tool (R) was used and a two-sided t-test with correction for multiple testing (BH) was performed to test for statistical significance.
Quantification of the mRNA of protein-coding genes differentially expressed in urt1sgi2 (a), cpl3sgi3 (b), and rst1sgi6 (c). Three biological repeats for each genotype were performed. A generalized linear model was applied to the whole dataset and the statistical significance was tested by pre-defined contrasts (mutant versus. ERF6-GR) using the glmfit function in EdgeR (R).
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Li, T., Natran, A., Chen, Y. et al. A genetics screen highlights emerging roles for CPL3, RST1 and URT1 in RNA metabolism and silencing. Nat. Plants 5, 539–550 (2019). https://doi.org/10.1038/s41477-019-0419-7
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