© Universität Bielefeld
Binding Landscape of AtGRP7
The lack of a genome-wide view on their in vivo binding targets and binding landscapes represents a gap in understanding the mode of action of plant RNA-binding proteins.
We have adapted individual nucleotide resolution crosslinking immunoprecipitation (iCLIP) (König et al, 2010, Nature Structural Molecular Biology) for genome-wide determining the binding repertoire RBPs, using the circadian clock-regulated Arabidopsis thaliana glycine-rich RNA-binding protein AtGRP7 as a paradigm.
The circadian transcriptome
iCLIP identified 858 transcripts with significantly enriched crosslink sites in plants expressing AtGRP7-GFP and 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.
452 of the iCLIP targets were also identified by RIP-seq, thus representing a set of high-confidence binders.
AtGRP7 can bind to all transcript regions with a preference for 3'untranslated regions.
The whole results of this studies were published in Meyer et al, 2017, Genome Biology.
Reports on our publication:
Establishing iCLIP for plants to identify target transcripts of the RNA-binding protein AtGRP7 paves the way to investigate the dynamics of posttranscriptional networks in response to exogenous and endogenous cues.