Abstract
RNA quality control (RQC) and post-transcriptional gene silencing (PTGS) target and degrade aberrant endogenous RNAs and foreign RNAs, contributing to homeostasis of cellular RNAs. In plants, RQC and PTGS compete for foreign and selected endogenous RNAs; however, little is known about the mechanism interconnecting the two pathways. Using a reporter system designed for monitoring PTGS, we revealed that the 26S proteasome subunit RPT2a enhances transgene PTGS by promoting the accumulation of transgene-derived short interfering RNAs without affecting their biogenesis. RPT2a physically associated with a subset of RQC components and downregulated the protein level. Overexpression of the RQC components interfered with transgene silencing, and impairment of the RQC machinery reinforced transgene PTGS attenuated by rpt2a. Overall, we demonstrate that the 26S proteasome subunit RPT2a promotes PTGS by repressing the RQC machinery to control foreign RNAs.
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Data availability
sRNA sequencing data were deposited to Gene Expression Omnibus with accession no. GSE131009. All data needed to evaluate the conclusions in the paper are present in the paper and/or the supplementary materials. Additional data related to this paper may be requested from the authors. Source data for Figs. 1–4 and Extended Data Figs. 4, 6 and 8 are provided with the paper.
References
Liu, L. & Chen, X. M. RNA quality control as a key to suppressing RNA silencing of endogenous genes in plants. Mol. Plant 9, 826–836 (2016).
Schwab, R. & Voinnet, O. RNA silencing amplification in plants: size matters. Proc. Natl Acad. Sci. USA 107, 14945–14946 (2010).
Schoenberg, D. R. & Maquat, L. E. Regulation of cytoplasmic mRNA decay. Nat. Rev. Genet. 13, 246–259 (2012).
Tsuzuki, M., Motomura, K., Kumakura, N. & Takeda, A. Interconnections between mRNA degradation and RDR-dependent siRNA production in mRNA turnover in plants. J. Plant Res. 130, 211–226 (2017).
Martinez 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).
Szadeczky-Kardoss, I. et al. The nonstop decay and the RNA silencing systems operate cooperatively in plants. Nucleic Acids Res. 46, 4632–4648 (2018).
Zhang, X. et al. Suppression of endogenous gene silencing by bidirectional cytoplasmic RNA decay in Arabidopsis. Science 348, 120–123 (2015).
Yu, A. et al. Second-site mutagenesis of a hypomorphic argonaute1 allele identifies SUPERKILLER3 as an endogenous suppressor of transgene posttranscriptional gene silencing. Plant Physiol. 169, 1266–1274 (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).
Zhao, L. & Kunst, L. SUPERKILLER complex components are required for the RNA exosome-mediated control of cuticular wax biosynthesis in Arabidopsis inflorescence stems. Plant Physiol. 171, 960–973 (2016).
Collins, G. A. & Goldberg, A. L. The logic of the 26S proteasome. Cell 169, 792–806 (2017).
Finley, D. Recognition and processing of ubiquitin–protein conjugates by the proteasome. Annu. Rev. Biochem. 78, 477–513 (2009).
Groll, M. et al. Structure of 20S proteasome from yeast at 2.4 A resolution. Nature 386, 463–471 (1997).
Finley, D., Chen, X. & Walters, K. J. Gates, channels, and switches: elements of the proteasome machine. Trends Biochem. Sci. 41, 77–93 (2016).
Vierstra, R. D. The ubiquitin–26S proteasome system at the nexus of plant biology. Nat. Rev. Mol. Cell Biol. 10, 385–397 (2009).
Lee, K. H. et al. The RPT2 subunit of the 26S proteasome directs complex assembly, histone dynamics, and gametophyte and sporophyte development in Arabidopsis. Plant Cell 23, 4298–4317 (2011).
Smalle, J. et al. The pleiotropic role of the 26S proteasome subunit RPN10 in Arabidopsis growth and development supports a substrate-specific function in abscisic acid signaling. Plant Cell 15, 965–980 (2003).
Smalle, J. et al. Cytokinin growth responses in Arabidopsis involve the 26S proteasome subunit RPN12. Plant Cell 14, 17–32 (2002).
Stadler, R. & Sauer, N. The Arabidopsis thaliana AtSUC2 gene is specifically expressed in companion cells. Bot. Acta 109, 299–306 (1996).
Carbonell, A. et al. New generation of artificial microRNA and synthetic trans-acting small interfering RNA vectors for efficient gene silencing in Arabidopsis. Plant Physiol. 165, 15–29 (2014).
Nadeau, J. A. & Sack, F. D. Control of stomatal distribution on the Arabidopsis leaf surface. Science 296, 1697–1700 (2002).
Baumberger, N. & Baulcombe, D. C. Arabidopsis ARGONAUTE1 is an RNA slicer that selectively recruits rnicroRNAs and short interfering RNAs. Proc. Natl Acad. Sci. USA 102, 11928–11933 (2005).
Ma, Z. & Zhang, X. Actions of plant Argonautes: predictable or unpredictable? Curr. Opin. Plant Biol. 45, 59–67 (2018).
Mourrain, P. et al. Arabidopsis SGS2 and SGS3 genes are required for posttranscriptional gene silencing and natural virus resistance. Cell 101, 533–542 (2000).
Dalmay, T., Hamilton, A., Rudd, S., Angell, S. & Baulcombe, D. C. An RNA-dependent RNA polymerase gene in Arabidopsis is required for posttranscriptional gene silencing mediated by a transgene but not by a virus. Cell 101, 543–553 (2000).
Baulcombe, D. RNA silencing in plants. Nature 431, 356–363 (2004).
Law, J. A. & Jacobsen, S. E. Establishing, maintaining and modifying DNA methylation patterns in plants and animals. Nat. Rev. Genet. 11, 204–220 (2010).
Xie, M. & Yu, B. siRNA-directed DNA methylation in plants. Curr. Genomics 16, 23–31 (2015).
Sonoda, Y. et al. Regulation of leaf organ size by the Arabidopsis RPT2a 19S proteasome subunit. Plant J. 60, 68–78 (2009).
Sijacic, P., Wang, W. & Liu, Z. Recessive antimorphic alleles overcome functionally redundant loci to reveal TSO1 function in Arabidopsis flowers and meristems. PLoS Genet. 7, e1002352 (2011).
Parent, J. S. et al. Post-transcriptional gene silencing triggered by sense transgenes involves uncapped antisense RNA and differs from silencing intentionally triggered by antisense transgenes. Nucleic Acids Res. 43, 8464–8475 (2015).
Smith, L. M. et al. An SNF2 protein associated with nuclear RNA silencing and the spread of a silencing signal between cells in Arabidopsis. Plant Cell 19, 1507–1521 (2007).
Martinez de Alba, A. E., Elvira-Matelot, E. & Vaucheret, H. Gene silencing in plants: a diversity of pathways. Biochim. Biophys. Acta 1829, 1300–1308 (2013).
Miki, D. et al. Efficient generation of diRNAs requires components in the posttranscriptional gene silencing pathway. Sci. Rep. 7, 301 (2017).
Motomura, K. et al. The role of decapping proteins in the miRNA accumulation in Arabidopsis thaliana. RNA Biol. 9, 644–652 (2012).
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).
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).
Shin, J. H. et al. The role of the Arabidopsis exosome in siRNA-independent silencing of heterochromatic loci. PLoS Genet. 9, e1003411 (2013).
Lam, P. et al. The exosome and trans-acting small interfering RNAs regulate cuticular wax biosynthesis during Arabidopsis inflorescence stem development. Plant Physiol. 167, 323–336 (2015).
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).
Erickson, S. L. et al. Competition between decapping complex formation and ubiquitin-mediated proteasomal degradation controls human Dcp2 decapping activity. Mol. Cell Biol. 35, 2144–2153 (2015).
Kelly, S. P. & Bedwell, D. M. Both the autophagy and proteasomal pathways facilitate the Ubp3p-dependent depletion of a subset of translation and RNA turnover factors during nitrogen starvation in Saccharomyces cerevisiae. RNA 21, 898–910 (2015).
Stribinskis, V. & Ramos, K. S. Rpm2p, a protein subunit of mitochondrial RNase P, physically and genetically interacts with cytoplasmic processing bodies. Nucleic Acids Res. 35, 1301–1311 (2007).
Brengues, M., Teixeira, D. & Parker, R. Movement of eukaryotic mRNAs between polysomes and cytoplasmic processing bodies. Science 310, 486–489 (2005).
Graham, A. C., Kiss, D. L. & Andrulis, E. D. Differential distribution of exosome subunits at the nuclear lamina and in cytoplasmic foci. Mol. Biol. Cell 17, 1399–1409 (2006).
Goldberg, A. L. Protein degradation and protection against misfolded or damaged proteins. Nature 426, 895–899 (2003).
Erales, J., Hoyt, M. A., Troll, F. & Coffino, P. Functional asymmetries of proteasome translocase pore. J. Biol. Chem. 287, 18535–18543 (2012).
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).
Sakamoto, T. et al. Arabidopsis thaliana 26S proteasome subunits RPT2a and RPT5a are crucial for zinc deficiency-tolerance. Biosci. Biotechnol. Biochem. 75, 561–567 (2011).
Sako, K. et al. Proteomic analysis of the 26S proteasome reveals its direct interaction with transit peptides of plastid protein precursors for their degradation. J. Proteome Res. 13, 3223–3230 (2014).
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).
Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics 25, 1754–1760 (2009).
McKenna, A. et al. The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 20, 1297–1303 (2010).
Martin, M. CUTADAPT removes adapter sequences from high-throughput sequencing reads. EMBnet J. 17, 10–12 (2011).
Johnson, N. R., Yeoh, J. M., Coruh, C. & Axtell, M. J. Improved placement of multi-mapping small RNAs. G3 6, 2103–2111 (2016).
Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).
Robinson, M. D., McCarthy, D. J. & Smyth, G. K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010).
Benjamini, Y. & Hochberg, Y. Controlling the false discovery rate—a practical and powerful approach to multiple testing. J. R. Stat. Soc. B 57, 289–300 (1995).
Langmead, B., Trapnell, C., Pop, M. & Salzberg, S. L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).
Wu, H. W., Lin, S. S., Chen, K. C., Yeh, S. D. & Chua, N. H. Discriminating mutations of HC-Pro of zucchini yellow mosaic virus with differential effects on small RNA pathways involved in viral pathogenicity and symptom development. Mol. Plant Microbe Interact. 23, 17–28 (2010).
Acknowledgements
We thank Y. Watanabe for the pDCP2-DCP2-GFP transgenic line, H. Lange for the RRP41-GFP and RRP41-MYC transgenic plants, H. Vaucheret for the L1 line, S. Park for the JAP3 transgenic plants, C. You and J. H. Park for the technical advice. This work was supported by IBS-R013-G2 from the Institute for Basic Science and in part by a grant from National Research Foundation (2019R1A2C3007376) and start-up funds from DGIST to J.M.K., and in part by (IBS‐R013‐D1) from the Institute for Basic Science.
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Y.J.K. and J.M.K. conceived the study and designed the experiments. M.-H.K., J.J., S.L. and J.M.P. performed the experiments. J.H.L. and L.G. analysed WGS and sRNA sequencing data. B.-H.L., Y.J.K. and J.M.K. wrote the manuscript.
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Peer review information Nature Plants thanks Xiuren Zhang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Extended data
Extended Data Fig. 1 Phenotypes of PORI plants.
(a) amiR-Luc sequence targeting GFPLuc. (b) Spatiotemporal GFP expression patterns in the prey, the Orion and the PORI plants. The cell type-specific expression of the TMM and SUC2 promoters were examined in transgenic plants harbouring the pTMM::GFP or pSUC2::GUS constructs. The arrowheads indicate a developing leaf where the GFP or the GUS expression is initiated and then disappeared or maintained, respectively. Scale bar, 1 mm. At least ten independent plants showed similar results. (c) Northern blot analysis of the accumulation of amiR-Luc in WT, prey, and PORI plants. U6 RNA was used as a loading control. The experiment was independently repeated four times with similar results.
Extended Data Fig. 2 Production of amiR-Luc in PORI plants is not regulated by the transcriptional gene silencing machinery.
(a) Northern blot analysis of amiR-Luc in the PORI and PORI/se-1, PORI/hyl-1, PORI/rdr6-11, and PORI/ago1-27. U6 RNA was used as a loading control. The experiment was independently repeated three times with similar results. (b) The size distribution of GFPLuc-siRNAs in the PORI plants. Data represent the mean of two independent replicates. (c) Luciferase activities in WT, LUCH, and PORI seedlings at 5 DAG in the absence or presence of 5-aza-dC. LUCH seedlings were used as a positive control. The experiment was independently repeated three times with similar results.
Extended Data Fig. 3 RPT2a plays a role in the suppression of GFP in PORI plants.
(a) Morphological phenotypes of rosette leaves, inflorescence, and size of Col, rpt2a-2, and rpt2a-6 plants. Scale bar, 1 cm. The segregation analysis of progenies from the genetic crosses is shown in Supplementary Table 1. (b) The detection of transiently expressed RPT2a and RPT2aΔ72bp in tobacco by western blot analysis. The experiment was repeated two times with similar results. (c) Co-immunoprecipitation analysis shows the interaction between RPT2a/RPT2aΔ72bp and RPT4a. RPT2a-flag, RPT2aΔ72bp-flag, and myc-RPT4a were expressed in tobacco plants. The experiment was repeated two times with similar results. (d) Allelic test among PORI, PORI/rpt2a-2 and PORI/rpt2a-6 plants. GFP fluorescence and morphological phenotypes of F1 plants. Scale bar, 500 µm (GFP), 1 cm (plants). The segregation analysis of progenies from the genetic crosses is shown in Supplementary Table 1. (e, f) Complementation test of PORI/rpt2a-6 by expressing p35S::RPT2a or pRPT2a::RPT2a. GFP fluorescence (e) and morphological phenotypes (f) of transgenic plants carrying p35S::RPT2a or pRPT2a::RPT2a in PORI/rpt2a-6. Scale bar, 500 µm (e), 10 cm (f). At least five independent transgenic plants showed similar results.
Extended Data Fig. 4 RPT2a promotes S-PTGS but not IR-PTGS.
(a, b) qRT-PCR analyses of GFPLuc (a) and TMM transcripts (b) in PORI and PORI/rpt2-6 plants. (c) GFP expression patterns of PORI, PORI/rpt2a-6, PORI/se-1, and PORI/rdr6-11 plants. Scale bar, 600 µm. The segregation analysis of progenies from the genetic crosses is shown in Supplementary Table 1. (d) qRT-PCR analysis GUS transcripts in L1 and L1/rpt2a-6 plants. (a, b, d) The expression levels were normalized to UBQ5 (At3g62250). Error bars represent mean ± s.d. from three independent biological replicates. (e) Photobleaching phenotypes in the IR-PTGS reporter JAP3 plants and JAP3/rpt2a-6. Scale bar, 1 cm. The segregation analysis of progenies from the genetic crosses is shown in Supplementary Table 1. (f) GFP fluorescence in the PORI plants treated with and without MG132. Scale bar, 1 mm. The experiment was independently repeated at least three times with similar results.
Extended Data Fig. 5 In vivo interaction between RPT2a and a subset of the RQC components, DCP2, RRP41 and RRP45a.
VPS9a (AT3G19770) (a), DCP1 (b) and RRP4 (c) were used as negative controls. RPT2a-GFP, RRP41-mCherry, RRP45a-mCherry, DCP2-mCherry, DCP1-mCherry, RRP4-mCherry, and VPS9a-mCherry were expressed in tobacco plants expressing SGS3-CFP. The experiment was independently repeated at least two times with similar results.
Extended Data Fig. 6 Transcript and protein levels of DCP2 and RRP45a in rpt2a-6.
(a) qRT-PCR analysis reveals the expression level of DCP2 in WT and rpt2a-6 plants normalized to UBQ5. Error bars represent mean ± s.d. calculated from three biological replicates. (b) Western blot analysis of RRP45a proteins in WT and rpt2a-6. The experiment was independently repeated three times with similar results.
Extended Data Fig. 7 RPT2a is translocated to cytoplasmic speckles in presence of SGS3.
(a) Subcellular localization of RPT2a, RRP41, and RRP45a. (b–d) Subcellular localization of RPT2a with DCP2 (b), RRP41 (c), and RRP45a (d) in the absence or presence of SGS3. The inset is an enlarged view of the region in the white box, in which a subset of co-localized granules of RPT2a and SGS3 are present in combination with DCP2, RRP41, or RRP45a. The proteins were transiently expressed in tobacco leaves. Scale bar, 20 µm (a), 50 µm (b–d). The experiment was independently repeated at least five times with similar results.
Extended Data Fig. 8 Transcript levels of DCP2 and RRP45a in 35s-DCP2 and 35s-RRP45a transgenic plants.
qRT-PCR reveals the expression levels of DCP2 and RRP45a in p35s-DCP2 and p35s-RRP45a transgenic plants. The relative expression values were obtained by normalization to UBQ5. Error bars represent mean ± s.d. calculated from three independent biological replicates.
Supplementary information
Supplementary Table 1
Segregation analysis of progenies from genetic crosses.
Supplementary Table 2
Primers used for plasmid construction.
Supplementary Table 3
Oligonucleotides used for RT–PCR and northern blot analysis.
Supplementary Table 4
Differentially expressed miRNAs in rpt2a-6.
Supplementary Table 5
Differentially expressed ta-siRNAs in rpt2a-6.
Supplementary Table 6
Differentially expressed hc-siRNAs in rpt2a-6.
Supplementary Table 7
Gene loci showing reduced levels of 21 nt sRNAs in rpt2a-6.
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Kim, MH., Jeon, J., Lee, S. et al. Proteasome subunit RPT2a promotes PTGS through repressing RNA quality control in Arabidopsis. Nat. Plants 5, 1273–1282 (2019). https://doi.org/10.1038/s41477-019-0546-1
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DOI: https://doi.org/10.1038/s41477-019-0546-1
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