Adaptation of iCLIP to plants determines the binding landscape of the clock-regulated RNA-binding protein AtGRP7

Meyer K, Köster T, Nolte C, Weinholdt C, Lewinski M, Grosse I, Staiger D (2017)
Genome Biology 18(1): 204.

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Journal Article | Original Article | Published | English
Abstract
Background Functions for RNA-binding proteins in orchestrating plant development and environmental responses are well established. However, the lack of a genome-wide view of their in vivo binding targets and binding landscapes represents a gap in understanding the mode of action of plant RNA-binding proteins. Here, we adapt individual nucleotide resolution crosslinking and immunoprecipitation (iCLIP) genome-wide to determine the binding repertoire of the circadian clock-regulated Arabidopsis thaliana glycine-rich RNA-binding protein AtGRP7. Results iCLIP identifies 858 transcripts with significantly enriched crosslink sites in plants expressing AtGRP7-GFP that are absent in plants expressing an RNA-binding-dead AtGRP7 variant or GFP alone. To independently validate the targets, we performed RNA immunoprecipitation (RIP)-sequencing of AtGRP7-GFP plants subjected to formaldehyde fixation. Of the iCLIP targets, 452 were also identified by RIP-seq and represent a set of high-confidence binders. AtGRP7 can bind to all transcript regions, with a preference for 3′ untranslated regions. In the vicinity of crosslink sites, U/C-rich motifs are overrepresented. Cross-referencing the targets against transcriptome changes in AtGRP7 loss-of-function mutants or AtGRP7-overexpressing plants reveals a predominantly negative effect of AtGRP7 on its targets. In particular, elevated AtGRP7 levels lead to damping of circadian oscillations of transcripts, including DORMANCY/AUXIN ASSOCIATED FAMILY PROTEIN2 and CCR-LIKE. Furthermore, several targets show changes in alternative splicing or polyadenylation in response to altered AtGRP7 levels. Conclusions We have established iCLIP for plants to identify target transcripts of the RNA-binding protein AtGRP7. This paves the way to investigate the dynamics of posttranscriptional networks in response to exogenous and endogenous cues.
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Meyer K, Köster T, Nolte C, et al. Adaptation of iCLIP to plants determines the binding landscape of the clock-regulated RNA-binding protein AtGRP7. Genome Biology. 2017;18(1): 204.
Meyer, K., Köster, T., Nolte, C., Weinholdt, C., Lewinski, M., Grosse, I., & Staiger, D. (2017). Adaptation of iCLIP to plants determines the binding landscape of the clock-regulated RNA-binding protein AtGRP7. Genome Biology, 18(1), 204. doi:10.1186/s13059-017-1332-x
Meyer, K., Köster, T., Nolte, C., Weinholdt, C., Lewinski, M., Grosse, I., and Staiger, D. (2017). Adaptation of iCLIP to plants determines the binding landscape of the clock-regulated RNA-binding protein AtGRP7. Genome Biology 18:204.
Meyer, K., et al., 2017. Adaptation of iCLIP to plants determines the binding landscape of the clock-regulated RNA-binding protein AtGRP7. Genome Biology, 18(1): 204.
K. Meyer, et al., “Adaptation of iCLIP to plants determines the binding landscape of the clock-regulated RNA-binding protein AtGRP7”, Genome Biology, vol. 18, 2017, : 204.
Meyer, K., Köster, T., Nolte, C., Weinholdt, C., Lewinski, M., Grosse, I., Staiger, D.: Adaptation of iCLIP to plants determines the binding landscape of the clock-regulated RNA-binding protein AtGRP7. Genome Biology. 18, : 204 (2017).
Meyer, Katja, Köster, Tino, Nolte, Christine, Weinholdt, Claus, Lewinski, Martin, Grosse, Ivo, and Staiger, Dorothee. “Adaptation of iCLIP to plants determines the binding landscape of the clock-regulated RNA-binding protein AtGRP7”. Genome Biology 18.1 (2017): 204.
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RNAs: dynamic and mutable.
Zavolan M, Graveley BR., Genome Biol 18(1), 2017
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