Proteasome subunit RPT2a promotes PTGS through repressing RNA quality control in Arabidopsis

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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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Fig. 1: GFPLuc in PORI plants undergoes miRNA- and siRNA-mediated PTGS.
Fig. 2: RPT2a is required for the production of transgene-derived siRNAs.
Fig. 3: Expression of endogenous miRNA and siRNA are not altered in rpt2a-6.
Fig. 4: A subset of RQC components is targets of RPT2a in vivo.

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.


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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.

Author information

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.

Correspondence to Yun Ju Kim or June M. Kwak.

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The authors declare no competing interests.

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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. Source data

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. Source data

Extended Data Fig. 7 RPT2a is translocated to cytoplasmic speckles in presence of SGS3.

(a) Subcellular localization of RPT2a, RRP41, and RRP45a. (bd) 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 (bd). 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. Source data

Supplementary information

Reporting Summary

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, M., Jeon, J., Lee, S. et al. Proteasome subunit RPT2a promotes PTGS through repressing RNA quality control in Arabidopsis. Nat. Plants 5, 1273–1282 (2019) doi:10.1038/s41477-019-0546-1

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