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

Abstract

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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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.

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Acknowledgements

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).

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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.

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Correspondence to Dirk Inzé.

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The authors declare no competing interests.

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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 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).

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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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