Article | Published:

RNA polymerase II activity revealed by GRO-seq and pNET-seq in Arabidopsis

Nature Plantsvolume 4pages11121123 (2018) | Download Citation


RNA polymerase II (Pol II) plays an essential role in gene expression. We used plant native elongating transcript sequencing and global run-on sequencing to profile nascent RNAs genome wide in Arabidopsis. We found that Pol II tends to accumulate downstream of the transcription start site (TSS). Moreover, Pol II with an unphosphorylated carboxyl-terminal domain (CTD) mainly accumulates downstream of the TSS, while Pol II with a Ser 5P CTD associates with spliceosomes, and Pol II with a Ser 2P CTD presents a sharp peak within 250 base pairs downstream of the polyadenylation site (PAS). Pol II pausing both at promoter-proximal regions and after PAS affects the transcription rate. Interestingly, active genes can be classified into three clusters based on the different modes of transcription. We demonstrate that these two methods are suitable to study Pol II dynamics in planta. Although transcription is conserved overall within eukaryotes, there is plant-specific regulation.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Data availability

The sequencing data and processed files are available at the Gene Expression Omnibus under accession numbers GSE109974 and GSE117014.

Additional information

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


  1. 1.

    Core, L. J., Waterfall, J. J. & Lis, J. T. Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters. Science 322, 1845–1848 (2008).

  2. 2.

    Kwak, H., Fuda, N. J., Core, L. J. & Lis, J. T. Precise maps of RNA polymerase reveal how promoters direct initiation and pausing. Science 339, 950–953 (2013).

  3. 3.

    Churchman, L. S. & Weissman, J. S. Nascent transcript sequencing visualizes transcription at nucleotide resolution. Nature 469, 368–373 (2011).

  4. 4.

    Rabani, M. et al. Metabolic labeling of RNA uncovers principles of RNA production and degradation dynamics in mammalian cells. Nat. Biotechnol. 29, 436–442 (2011).

  5. 5.

    Schwalb, B. et al. TT-seq maps the human transient transcriptome. Science 352, 1225–1228 (2016).

  6. 6.

    Harlen, K. M. & Churchman, L. S. The code and beyond: transcription regulation by the RNA polymerase II carboxy-terminal domain. Nat. Rev. Mol. Cell Biol. 18, 263–273 (2017).

  7. 7.

    Nojima, T. et al. Mammalian NET-Seq reveals genome-wide nascent transcription coupled to RNA processing. Cell 161, 526–540 (2015).

  8. 8.

    Zhu, J.-K. Abiotic stress signaling and responses in plants. Cell 167, 313–324 (2016).

  9. 9.

    Singh, K. B., Foley, R. C. & Oñate-Sánchez, L. Transcription factors in plant defense and stress responses. Curr. Opin. Plant. Biol. 5, 430–436 (2002).

  10. 10.

    Erhard, K. F., Talbot, J.-E. R. B., Deans, N. C., McClish, A. E. & Hollick, J. B. Nascent transcription affected by RNA polymerase IV in Zea mays. Genetics 199, 1107–1125 (2015).

  11. 11.

    Hetzel, J., Duttke, S. H., Benner, C. & Chory, J. Nascent RNA sequencing reveals distinct features in plant transcription. Proc. Natl Acad. Sci. USA 113, 12316–12321 (2016).

  12. 12.

    Liu, W. et al. RNA-directed DNA methylation involves co-transcriptional small-RNA-guided slicing of polymerase V transcripts in Arabidopsis. Nat. Plants 4, 181–188 (2018).

  13. 13.

    Liu, C. et al. Arabidopsis ARGONAUTE 1 binds chromatin to promote gene transcription in response to hormones and stresses. Dev. Cell 44, 1–14 (2018).

  14. 14.

    Hawley, D. K. & Roeder, R. G. Separation and partial characterization of three functional steps in transcription initiation by human RNA polymerase II. J. Biol. Chem. 260, 8163–8172 (1985).

  15. 15.

    Rougvie, A. E. & Lis, J. T. The RNA polymerase II molecule at the 5′ end of the uninduced hsp70 gene of D. melanogaster is transcriptionally engaged. Cell 54, 795–804 (1988).

  16. 16.

    Folta, K. M. & Kaufman, L. S. Isolation of Arabidopsis nuclei and measurement of gene transcription rates using nuclear run-on assays. Nat. Protoc. 1, 3094–3100 (2006).

  17. 17.

    Core, LeightonJ. et al. Defining the status of RNA polymerase at promoters. Cell Reports 2, 1025–1035 (2012).

  18. 18.

    Schlackow, M. et al. Distinctive patterns of transcription and RNA processing for human lincRNAs. Mol. Cell 65, 25–38 (2017).

  19. 19.

    Liu, Y. et al. PCSD: a plant chromatin state database. Nucleic Acids Res. 46, D1157–D1167 (2018).

  20. 20.

    Hajheidari, M., Koncz, C. & Eick, D. Emerging roles for RNA polymerase II CTD in Arabidopsis. Trends Plant Sci. 18, 633–643 (2013).

  21. 21.

    Zhang, B. et al. C-terminal domain (CTD) phosphatase links Rho GTPase signaling to Pol II CTD phosphorylation in Arabidopsis and yeast. Proc. Natl Acad. Sci. USA 113, E8197–E8206 (2016).

  22. 22.

    Lin, J., Xu, R., Wu, X., Shen, Y. & Li, Q. Q. Role of cleavage and polyadenylation specificity factor 100: anchoring poly(A) sites and modulating transcription termination. Plant J. 91, 829–839 (2017).

  23. 23.

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

  24. 24.

    Li, G. et al. ISWI proteins participate in the genome-wide nucleosome distribution in Arabidopsis. Plant J. 78, 706–714 (2014).

  25. 25.

    Wang, D. et al. Reprogramming transcription by distinct classes of enhancers functionally defined by eRNA. Nature 474, 390–394 (2011).

  26. 26.

    Martin, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J. 17, 10–12 (2011).

  27. 27.

    Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).

  28. 28.

    Kurtz, S., Narechania, A., Stein, J. C. & Ware, D. A new method to compute K-mer frequencies and its application to annotate large repetitive plant genomes. BMC Genom. 9, 517 (2008).

  29. 29.

    Bushnell, B. BBMap: A Fast, Accurate, Splice-aware Aligner Report No. LBNL-7065E (Lawrence Berkeley National Laboratory, 2014).

  30. 30.

    Kolde, R. pheatmap: Pretty Heatmaps. R package version 1.0.8 (2015).

  31. 31.

    Hennig, C. fpc: Flexible Procedures for Clustering. R package version 2.1-10 (2015).

  32. 32.

    Yu, G., Wang, L.-G., Han, Y. & He, Q.-Y. clusterProfiler: an R package for comparing biological themes among gene clusters. OMICS 16, 284–287 (2012).

  33. 33.

    Quinlan, A. R. & Hall, I. M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010).

  34. 34.

    Kim, D. et al. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 14, R36 (2013).

  35. 35.

    Anders, S., Pyl, P. T. & Huber, W. HTSeq-a Python framework to work with high-throughput sequencing data. Bioinformatics 31, 166–169 (2015).

Download references


This work was supported by the General Programme of the National Natural Science Foundation of China (grant numbers 31471165 and 31271318) and the Key Laboratory of Plant Resource Conservation and Sustainable Utilization, Chinese Academy of Sciences, to Z.D.

Author information

Author notes

  1. These authors contributed equally: Jiafu Zhu and Min Liu.


  1. Plant Gene Engineering Centre, Key Laboratory of South China Agricultural Plant Molecular Analysis and Genetic Improvement, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, China

    • Jiafu Zhu
    • , Min Liu
    • , Xiaobin Liu
    •  & Zhicheng Dong
  2. University of Chinese Academy of Sciences, Beijing, China

    • Jiafu Zhu
  3. School of Life Sciences, Guangzhou University, Guangzhou Higher Education Mega Center, Guangzhou, China

    • Min Liu
    •  & Zhicheng Dong
  4. Center for Ecological and Environmental Sciences, Northwestern Polytechnical University, Xi’an, China

    • Min Liu


  1. Search for Jiafu Zhu in:

  2. Search for Min Liu in:

  3. Search for Xiaobin Liu in:

  4. Search for Zhicheng Dong in:


Z.D. designed the research. J.Z. and X.L. performed experiments. M.L. analysed data. Z.D., M.L. and J.Z. wrote the manuscript.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Zhicheng Dong.

Supplementary information

  1. Supplementary Information

    Supplementary Figures 1–12 and Supplementary Tables 1–4.

  2. Reporting Summary

About this article

Publication history