A genetics screen highlights emerging roles for CPL3, RST1 and URT1 in RNA metabolism and silencing


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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1: Characteristics of seven identified EMS mutants and validation of the causative gene.
Fig. 2: Transgene transcript degradation in urt1sgi2, cpl3sgi3 and rst1sgi6.
Fig. 3: Transgene-derived siRNA accumulation in urt1sgi2, cpl3sgi3 and rst1sgi6.
Fig. 4: Effect of mutation of URT1, CPL3 and RST1 on endogenous gene expression, siRNAs and plant development.
Fig. 5: Model for URT1-, CPL3- and RST1-mediated PTGS.

Data availability

The data that support the findings of this study are available from the corresponding author upon request.


  1. 1.

    Baulcombe, D. RNA silencing in plants. Nature 431, 356–363 (2004).

    CAS  Article  Google Scholar 

  2. 2.

    Chen, X. A silencing safeguard: links between RNA silencing and mRNA processing in Arabidopsis. Dev. Cell 14, 811–812 (2008).

    CAS  Article  Google Scholar 

  3. 3.

    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).

    CAS  Article  Google Scholar 

  4. 4.

    Hentze, M. W. & Kulozik, A. E. A perfect message: RNA surveillance and nonsense-mediated decay. Cell 96, 307–310 (1999).

    CAS  Article  Google Scholar 

  5. 5.

    Belostotsky, D. mRNA turnover meets RNA interference. Mol. Cell 16, 498–500 (2004).

    CAS  Article  Google Scholar 

  6. 6.

    Parker, R. & Song, H. The enzymes and control of eukaryotic mRNA turnover. Nat. Struct. Mol. Biol. 11, 121–127 (2004).

    CAS  Article  Google Scholar 

  7. 7.

    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).

    CAS  Article  Google Scholar 

  8. 8.

    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).

    CAS  Article  Google Scholar 

  9. 9.

    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).

    CAS  Article  Google Scholar 

  10. 10.

    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).

    CAS  Article  Google Scholar 

  11. 11.

    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).

    CAS  Article  Google Scholar 

  12. 12.

    Dorcey, E. et al. Context-dependent dual role of SKI8 homologs in mRNA synthesis and turnover. PLoS Genet. 8, e1002652 (2012).

    CAS  Article  Google Scholar 

  13. 13.

    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).

    CAS  Article  Google Scholar 

  14. 14.

    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).

    CAS  Article  Google Scholar 

  15. 15.

    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).

    CAS  Article  Google Scholar 

  16. 16.

    Zhang, X. et al. Suppression of endogenous gene silencing by bidirectional cytoplasmic RNA decay in Arabidopsis. Science 348, 120–123 (2015).

    CAS  Article  Google Scholar 

  17. 17.

    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).

    CAS  Article  Google Scholar 

  18. 18.

    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).

    CAS  Article  Google Scholar 

  19. 19.

    Kumakura, N. et al. SGS3 and RDR6 interact and colocalize in cytoplasmic SGS3/RDR6‐bodies. FEBS Lett. 583, 1261–1266 (2009).

    CAS  Article  Google Scholar 

  20. 20.

    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).

    CAS  Article  Google Scholar 

  21. 21.

    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).

    CAS  Article  Google Scholar 

  22. 22.

    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).

    CAS  Article  Google Scholar 

  23. 23.

    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).

    Article  Google Scholar 

  24. 24.

    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).

    Article  Google Scholar 

  25. 25.

    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).

    CAS  Google Scholar 

  26. 26.

    Stepien, A. et al. Posttranscriptional coordination of splicing and miRNA biogenesis in plants. RNA 8, e1403 (2017).

    Google Scholar 

  27. 27.

    Bartel, D. P. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281–297 (2004).

    CAS  Article  Google Scholar 

  28. 28.

    Bologna, N. G. et al. Nucleo-cytosolic shuttling of ARGONAUTE1 prompts a revised model of the plantmicroRNA pathway. Mol. Cell 69, 709–719 (2018).

    CAS  Article  Google Scholar 

  29. 29.

    Bollman, K. M. et al. HASTY, the Arabidopsis ortholog of exportin 5/MSN5, regulates phase change and morphogenesis. Development 130, 1493–1504 (2003).

    CAS  Article  Google Scholar 

  30. 30.

    Li, S. et al. MicroRNAs inhibit the translation of target mRNAs on the endoplasmic reticulum in Arabidopsis. Cell 153, 562–574 (2013).

    CAS  Article  Google Scholar 

  31. 31.

    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).

    CAS  Article  Google Scholar 

  32. 32.

    Carbonell, A. et al. Functional analysis of three Arabidopsis ARGONAUTES using slicer-defective mutants. Plant Cell 24, 3613–3629 (2012).

    CAS  Article  Google Scholar 

  33. 33.

    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).

    CAS  Article  Google Scholar 

  34. 34.

    Tang, G. siRNA and miRNA: an insight into RISCs. Trends Biochem. Sci. 30, 106–114 (2005).

    CAS  Article  Google Scholar 

  35. 35.

    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).

    CAS  Article  Google Scholar 

  36. 36.

    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).

    Article  Google Scholar 

  37. 37.

    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).

    CAS  Article  Google Scholar 

  38. 38.

    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).

    Article  Google Scholar 

  39. 39.

    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).

    CAS  Article  Google Scholar 

  40. 40.

    Elmayan, T. et al. Arabidopsis mutants impaired in cosuppression. Plant Cell 10, 1747–1757 (1998).

    CAS  Article  Google Scholar 

  41. 41.

    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).

    CAS  Article  Google Scholar 

  42. 42.

    Sement, F. M. et al. Uridylation prevents 3′ trimming of oligoadenylated mRNAs. Nucleic Acids Res. 41, 7115–7127 (2013).

    CAS  Article  Google Scholar 

  43. 43.

    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).

    Google Scholar 

  44. 44.

    Lim, J. et al. Uridylation by TUT4 and TUT7 marks mRNA for degradation. Cell 159, 1365–1376 (2014).

    CAS  Article  Google Scholar 

  45. 45.

    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).

    Article  Google Scholar 

  46. 46.

    Li, F. et al. Modulation of RNA polymerase II phosphorylation downstream of pathogen perception orchestrates plant immunity. Cell Host Microbe 16, 748–758 (2014).

    CAS  Article  Google Scholar 

  47. 47.

    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).

    CAS  Article  Google Scholar 

  48. 48.

    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).

    Article  Google Scholar 

  49. 49.

    Manavella, P. A. et al. Fast-forward genetics identifies plant CPL phosphatases as regulators of miRNA processing factor HYL1. Cell 151, 859–870 (2012).

    CAS  Article  Google Scholar 

  50. 50.

    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).

    CAS  Article  Google Scholar 

  51. 51.

    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).

    Article  Google Scholar 

  52. 52.

    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).

    CAS  Article  Google Scholar 

  53. 53.

    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).

    CAS  Article  Google Scholar 

  54. 54.

    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).

    CAS  Article  Google Scholar 

  55. 55.

    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).

    CAS  Article  Google Scholar 

  56. 56.

    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).

    CAS  Article  Google Scholar 

  57. 57.

    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).

    CAS  Article  Google Scholar 

  58. 58.

    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).

    Article  Google Scholar 

  59. 59.

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  60. 60.

    Telfer, A. & Poethig, R. S. HASTY: a gene that regulates the timing of shoot maturation in Arabidopsis thaliana. Development 125, 1889–1898 (1998).

    CAS  PubMed  Google Scholar 

  61. 61.

    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).

    CAS  Article  Google Scholar 

  62. 62.

    McElver, J. et al. Insertional mutagenesis of genes required for seed development in Arabidopsis thaliana. Genetics 159, 1751–1763 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  63. 63.

    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).

    CAS  Article  Google Scholar 

  64. 64.

    Schneeberger, K. et al. SHOREmap: simultaneous mapping and mutation identification by deep sequencing. Nat. Methods 6, 550–551 (2009).

    CAS  Article  Google Scholar 

  65. 65.

    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).

    CAS  Article  Google Scholar 

  66. 66.

    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).

    CAS  Article  Google Scholar 

  67. 67.

    Löw, R. in The Nucleic Acid Protocols Handbook (ed. Rapley, R.) 239–247 (Humana Press, 2000).

  68. 68.

    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).

    CAS  Article  Google Scholar 

  69. 69.

    Sement, F. M. & Gagliardi, D. Detection of uridylated mRNAs. Methods Mol. Biol. 1125, 43–51 (2014).

    CAS  Article  Google Scholar 

  70. 70.

    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).

  71. 71.

    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).

    CAS  Article  Google Scholar 

  72. 72.

    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).

    CAS  Article  Google Scholar 

Download references


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).

Author information




T.L., M.D., N.G. and D.I. designed the research. T.L. performed most of the experiments. M.D. performed the forwards genetics screen. A.N. assisted T.L. in finishing the experiments. M.D. analysed the DNA and mRNA sequencing data. Y.J.C and K.W. analysed the sRNA sequencing data. J.V. assisted in finalizing the manuscript. T.L., M.D., N.G. and D.I. wrote the article.

Corresponding author

Correspondence to Dirk Inzé.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

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.

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figures 1–10 and Supplementary Tables 1 and 4–6.

Reporting Summary

Supplementary Table 2

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.

Supplementary Table 3

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).

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

Further reading


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing