Adaptation of iCLIP to plants determines the binding landscape of the clock-regulated RNA-binding protein AtGRP7
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 RN...
| Main Authors: | , , , , , , |
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| Format: | Article |
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BMC
2017-10-01
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| Series: | Genome Biology |
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| Online Access: | http://link.springer.com/article/10.1186/s13059-017-1332-x |
| _version_ | 1818110533039030272 |
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| author | Katja Meyer Tino Köster Christine Nolte Claus Weinholdt Martin Lewinski Ivo Grosse Dorothee Staiger |
| author_facet | Katja Meyer Tino Köster Christine Nolte Claus Weinholdt Martin Lewinski Ivo Grosse Dorothee Staiger |
| author_sort | Katja Meyer |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-11T02:48:39Z |
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| institution | Directory Open Access Journal |
| issn | 1474-760X |
| language | English |
| last_indexed | 2024-12-11T02:48:39Z |
| publishDate | 2017-10-01 |
| publisher | BMC |
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| series | Genome Biology |
| spelling | doaj.art-56e0908ee67e415cb0ea98ac9a89dfa42022-12-22T01:23:21ZengBMCGenome Biology1474-760X2017-10-0118112210.1186/s13059-017-1332-xAdaptation of iCLIP to plants determines the binding landscape of the clock-regulated RNA-binding protein AtGRP7Katja Meyer0Tino Köster1Christine Nolte2Claus Weinholdt3Martin Lewinski4Ivo Grosse5Dorothee Staiger6RNA Biology and Molecular Physiology, Faculty of Biology, Bielefeld UniversityRNA Biology and Molecular Physiology, Faculty of Biology, Bielefeld UniversityRNA Biology and Molecular Physiology, Faculty of Biology, Bielefeld UniversityInstitute of Computer Science, Martin Luther University Halle-WittenbergRNA Biology and Molecular Physiology, Faculty of Biology, Bielefeld UniversityInstitute of Computer Science, Martin Luther University Halle-WittenbergRNA Biology and Molecular Physiology, Faculty of Biology, Bielefeld UniversityAbstract 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.http://link.springer.com/article/10.1186/s13059-017-1332-xCircadian rhythmIndividual nucleotide resolution crosslinking and immunoprecipitation (iCLIP)RNA immunoprecipitation (RIP)RNA-binding protein |
| spellingShingle | Katja Meyer Tino Köster Christine Nolte Claus Weinholdt Martin Lewinski Ivo Grosse Dorothee Staiger Adaptation of iCLIP to plants determines the binding landscape of the clock-regulated RNA-binding protein AtGRP7 Genome Biology Circadian rhythm Individual nucleotide resolution crosslinking and immunoprecipitation (iCLIP) RNA immunoprecipitation (RIP) RNA-binding protein |
| title | Adaptation of iCLIP to plants determines the binding landscape of the clock-regulated RNA-binding protein AtGRP7 |
| title_full | Adaptation of iCLIP to plants determines the binding landscape of the clock-regulated RNA-binding protein AtGRP7 |
| title_fullStr | Adaptation of iCLIP to plants determines the binding landscape of the clock-regulated RNA-binding protein AtGRP7 |
| title_full_unstemmed | Adaptation of iCLIP to plants determines the binding landscape of the clock-regulated RNA-binding protein AtGRP7 |
| title_short | Adaptation of iCLIP to plants determines the binding landscape of the clock-regulated RNA-binding protein AtGRP7 |
| title_sort | adaptation of iclip to plants determines the binding landscape of the clock regulated rna binding protein atgrp7 |
| topic | Circadian rhythm Individual nucleotide resolution crosslinking and immunoprecipitation (iCLIP) RNA immunoprecipitation (RIP) RNA-binding protein |
| url | http://link.springer.com/article/10.1186/s13059-017-1332-x |
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