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Post-transcriptional splicing of nascent RNA contributes to widespread intron retention in plants

Nature Plants volume 6, pages 780–788 (2020)Cite this article

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Abstract

In eukaryotes, genes are transcribed by RNA polymerase-II (Pol-II) and introns are removed by the spliceosome largely cotranscriptionally1,2,3; analysis using long-read sequencing revealed that splicing occurs immediately after Pol-II passes introns in yeast4,5. Here, we developed a Nanopore-based method to profile chromatin-bound RNA that enables the simultaneous detection of splicing status, Pol-II position and polyadenylation at the genome-wide scale in Arabidopsis. We found that more than half of the introns remain unspliced after Pol-II transcribes 1 kb past the 3′ splice site, which is much slower than the rate of splicing reported in yeast4,5. Many of the full-length chromatin-bound RNA molecules are polyadenylated, yet still contain unspliced introns at specific positions. These introns are nearly absent in the cytoplasm and are resistant to nonsense-mediated decay, suggesting that they are post-transcriptionally spliced before the transcripts are released into the cytoplasm; we therefore termed these introns post-transcriptionally spliced introns (pts introns). Analysis of around 6,500 public RNA-sequencing libraries found that the splicing of pts introns requires the function of splicing-related proteins such as PRMT5 and SKIP, and is also influenced by various environmental signals. The majority of the intron retention events in Arabidopsis are at pts introns, suggesting that chromatin-tethered post-transcriptional splicing is a major contributor to the widespread intron retention that is observed in plants, and could be a mechanism to produce fully spliced functional mRNAs for rapid response.

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Fig. 1: Full-length nascent RNA-seq.
Fig. 2: Splicing can occur cotranscriptionally in Arabidopsis and is coupled in multi-intron genes.
Fig. 3: High accumulation of polyadenylated transcripts with unspliced introns at specific positions on chromatin.
Fig. 4: The splicing of pts introns was regulated by splicing-related factors and various environmental signals.

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Data availability

All data generated in this study were deposited at NCBI under the accession number PRJNA591665.

Code availability

The code used to perform Poly(A) tail analysis is available at https://github.com/zhailab/polyACaller.

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Acknowledgements

We thank K.-H. Yeom and Z. Wu for advice on chromatin-bound RNA isolation. The group of J.Z. is supported by the National Key R&D Program of China Grant (2019YFA0903903); an NSFC to J.Z. (grant no. 31871234); the Program for Guangdong Introducing Innovative and Entrepreneurial Teams (2016ZT06S172); and the Shenzhen Sci-Tech Fund (KYTDPT20181011104005). J.J. is supported by China Postdoctoral Science Foundation (2018M640787). The group of X.C. is supported by the National Natural Science Foundation of China (grant nos. 31788103 and 91540203, to X.C.); the Chinese Academy of Sciences (Strategic Priority Research Program, XDB27030201 and QYZDY-SSW-SMC022, to X.C.); the Youth Innovation Promotion Association of CAS (grant no. 2018131, to X.D.); and the State Key Laboratory of Plant Genomics.

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Author notes

  1. These authors contributed equally: Jinbu Jia, Yanping Long.

Authors and Affiliations

  1. Institute of Plant and Food Science, Department of Biology, Southern University of Science and Technology, Shenzhen, China

    Jinbu Jia, Yanping Long, Hong Zhang, Zhuowen Li, Zhijian Liu, Yan Zhao, Dongdong Lu, Xianhao Jin & Jixian Zhai

  2. College of Horticulture, South China Agricultural University, Guangzhou, China

    Jinbu Jia & Rui Xia

  3. Institute for Advanced Studies and College of Life Science, Wuhan University, Wuhan, China

    Yanping Long

  4. State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China

    Xian Deng & Xiaofeng Cao

  5. University of Chinese Academy of Sciences, Beijing, China

    Xiaofeng Cao

  6. Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Beijing, China

    Xiaofeng Cao

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Contributions

J.J., Y.L., D.L., X.J. and X.D. performed the experiments. J.J., Y.L., H.Z., Z.Li, Z.Liu and Y.Z. analysed the data. R.X., X.C. and J.Z. oversaw the study. J.J., Y.L. and J.Z. wrote the manuscript, and all of the authors revised the manuscript.

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Correspondence to Jixian Zhai.

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Supplementary Information

Supplementary Figs. 1–11 and the unprocessed western blots for Supplementary Fig. 1.

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Jia, J., Long, Y., Zhang, H. et al. Post-transcriptional splicing of nascent RNA contributes to widespread intron retention in plants. Nat. Plants 6, 780–788 (2020). https://doi.org/10.1038/s41477-020-0688-1

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