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Mapping protein–RNA binding in plants with individual-nucleotide-resolution UV cross-linking and immunoprecipitation (plant iCLIP2)

Nature Protocols volume 19, pages 1183–1234 (2024)Cite this article

Subjects

Abstract

Despite crucial roles of RNA-binding proteins (RBPs) in plant physiology and development, methods for determining their transcriptome-wide binding landscape are less developed than those used in other model organisms. Cross-linking and immunoprecipitation (CLIP) methods (based on UV-mediated generation of covalent bonds between RNAs and cognate RBPs in vivo, purification of the cross-linked complexes and identification of the co-purified RNAs by high-throughput sequencing) have been applied mainly in mammalian cells growing in monolayers or in translucent tissue. We have developed plant iCLIP2, an efficient protocol for performing individual-nucleotide-resolution CLIP (iCLIP) in plants, tailored to overcome the experimental hurdles posed by plant tissue. We optimized the UV dosage to efficiently cross-link RNA and proteins in plants and expressed epitope-tagged RBPs under the control of their native promoters in loss-of-function mutants. We select epitopes for which nanobodies are available, allowing stringent conditions for immunopurification of the RNA–protein complexes to be established. To overcome the inherently high RNase content of plant cells, RNase inhibitors are added and the limited RNA fragmentation step is modified. We combine the optimized isolation of RBP-bound RNAs with iCLIP2, a streamlined protocol that greatly enhances the efficiency of library preparation for high-throughput sequencing. Plant researchers with experience in molecular biology and handling of RNA can complete this iCLIP2 protocol in ~5 d. Finally, we describe a bioinformatics workflow to determine targets of Arabidopsis RBPs from iCLIP data, covering all steps from downloading sequencing reads to identifying cross-linking events (https://github.com/malewins/Plant-iCLIPseq), and present the R/Bioconductor package BindingSiteFinder to extract reproducible binding sites (https://bioconductor.org/packages/release/bioc/html/BindingSiteFinder.html).

Key points

  • As in mammals, RBPs in plants are key regulators of the RNA life cycle. This protocol describes an optimized plant iCLIP method to define the transcriptome-wide RBP binding landscape at single-nucleotide resolution.

  • This plant iCLIP protocol, entailing UV cross-linking and nanobody-mediated precipitation of tagged RBP–RNA complexes, is optimized for efficient library preparation. A streamlined bioinformatics pipeline is also provided for the identification of RBP binding sites.

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Fig. 1: A schematic of the plant iCLIP2 procedure.
Fig. 2: A schematic outline of the bioinformatics workflow to obtain binding sites from Arabidopsis iCLIP data.
Fig. 3: Optimization of cross-linking, control IPs and RNase digestion.
Fig. 4: Timing of the plant iCLIP2 wet lab protocol.
Fig. 5: Western analysis of proteins recovered by IP.
Fig. 6: Oligonucleotides used in the individual steps of iCLIP2 library generation.
Fig. 7: Control for the efficiency of the first ProNex size selection.
Fig. 8: Second PCR (optimization PCR) and second ProNex size selection.
Fig. 9: Flow chart of the bioinformatics workflow to obtain binding sites from a sequenced plant iCLIP2 data.
Fig. 10: Script execution order for Arabidopsis iCLIP analysis with names and locations in the corresponding folder.
Fig. 11: Frequency of tetramers at the position of sample barcodes (positions 4–7).
Fig. 12: AtGRP7 iCLIP cross-links and reproducible binding sites on FLL2.
Fig. 13: Read quality and adapter content before and after processing.
Fig. 14: Representative results from binding site definition using the R/Bioconductor package BindingSiteFinder.

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Data availability

The AtGRP7 iCLIP dataset processed in the bioinformatics workflow is available through the Gene Expression Omnibus series GSE99427 or directly via SRA by the accession number SRR24391474. Source data are provided with this paper.

Code availability

All necessary code is publicly available under the Massachusetts Institute of Technology license. The R package BindingSiteFinder is accessible via the Bioconductor repository at https://bioconductor.org/packages/release/bioc/html/BindingSiteFinder.html. The scripts described in this publication are accessible via GitHub at https://github.com/malewins/Plant-iCLIPseq.

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Acknowledgements

We thank K. Neudorf and E. Klemme for expert technical assistance throughout the development of the procedure. Work in our laboratories is supported by the German Research Foundation through grants STA653/14-1, 15-1 and 19-1 to D.S., SPP 1935 ZA881/5-2 and FOR 2333 ZA881/3-1 to K.Z., SPP 1935 KO4566/3-2 and FOR 2333 KO4566/5-1 to J.K., and by a postdoctoral fellowship of the Alexander von Humboldt Foundation to M.R.

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Author notes

  1. These authors contributed equally: Martin Lewinski, Mirko Brüggemann, Tino Köster.

Authors and Affiliations

  1. RNA Biology and Molecular Physiology, Faculty of Biology, Bielefeld University, Bielefeld, Germany

    Martin Lewinski, Tino Köster, Marlene Reichel, Thorsten Bergelt, Katja Meyer & Dorothee Staiger

  2. Buchmann Institute for Molecular Life Sciences (BMLS) & Institute of Molecular Biosciences, Goethe University Frankfurt, Frankfurt, Germany

    Mirko Brüggemann & Kathi Zarnack

  3. Institute of Molecular Biology (IMB), Mainz, Germany

    Julian König

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Contributions

T.K., M.R. and K.M. developed the experimental methods. M.L. developed the processing part of the bioinformatics workflow. M.B. developed the R/Bioconductor package BindingSiteFinder. D.S. and J.K. directed the development of the experimental methods. K.Z. directed the development of the bioinformatics workflow. M.R. assembled part 1 of the procedure pertaining to the experimental protocol. M.L. and M.B. assembled part 2 of the procedure pertaining to the bioinformatics workflow. T.B. assembled the Materials section. K.M. produced the data in Fig. 2. D.S. wrote the manuscript with contributions from all authors.

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Correspondence to Dorothee Staiger.

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Key references using this protocol

Meyer, K. et al. Genome Biol. 18, 204 (2017): https://doi.org/10.1186/s13059-017-1332-x

Arribas-Hernandez, L. et al. eLife 10, e72375 (2021): https://doi.org/10.7554/eLife.72375

Buchbender, A. et al. Methods 178, 33–48 (2020): https://doi.org/10.1016/j.ymeth.2019.10.003

Busch, A. et al. Methods 178, 49–62 (2020): https://doi.org/10.1016/j.ymeth.2019.11.008

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Unprocessed gels.

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Lewinski, M., Brüggemann, M., Köster, T. et al. Mapping protein–RNA binding in plants with individual-nucleotide-resolution UV cross-linking and immunoprecipitation (plant iCLIP2). Nat Protoc 19, 1183–1234 (2024). https://doi.org/10.1038/s41596-023-00935-3

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