Schmitz-Linneweber C, Williams-Carrier R, Barkan A. Genomic era analyses of RNA secondary structure and RNA-binding proteins reveal their significance to post-transcriptional regulation in plants. Marker positions and the location of the AtGRP7-GFP RNA adducts are indicated Immunoblot against AtGRP7 identifies the precipitated protein ( bottom). c Autoradiogram of RNA–protein complexes of UV crosslinked AtGRP7::AtGRP7- GFP grp7-1 plants, AtGRP7::AtGRP7 R 49 Q- GFP, and AtGRP7::GFP-only plants. Treatment of the precipitate with RNase I ( + RNase) indicates the size of the precipitated protein. b Autoradiogram of RNA–protein complexes from AtGRP7::AtGRP7- GFP grp7-1 plants after UV XL and without UV XL and after precipitation ( IP+) or mock precipitation ( IP−). Positions of the molecular weight markers are indicated. For comparison, the membrane was stained with amidoblack. a Aliquots of the lysate (input, IN), IP+, IP− and the supernatant ( SN) of the precipitations were analyzed by immunoblotting with the α- AtGRP7 antibody. Lysates were subjected to immunoprecipitation with GFP Trap beads ( IP+) and mock precipitation with RFP Trap beads ( IP−). RNA–protein interactions were stabilized by UV irradiation of 16-day-old plants with UV light (254 nm) at 500 mJ/cm 2. Immunoprecipitation of AtGRP7 protein–RNA complexes from UV crosslinked AtGRP7::AtGRP7-GFP grp7-1 plants.

This paves the way to investigate the dynamics of posttranscriptional networks in response to exogenous and endogenous cues.Ĭircadian rhythm Individual nucleotide resolution crosslinking and immunoprecipitation (iCLIP) RNA immunoprecipitation (RIP) RNA-binding protein. We have established iCLIP for plants to identify target transcripts of the RNA-binding protein AtGRP7. Furthermore, several targets show changes in alternative splicing or polyadenylation in response to altered AtGRP7 levels. In particular, elevated AtGRP7 levels lead to damping of circadian oscillations of transcripts, including DORMANCY/AUXIN ASSOCIATED FAMILY PROTEIN2 and CCR-LIKE. 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 the vicinity of crosslink sites, U/C-rich motifs are overrepresented.

AtGRP7 can bind to all transcript regions, with a preference for 3' untranslated regions. Of the iCLIP targets, 452 were also identified by RIP-seq and represent a set of high-confidence binders. To independently validate the targets, we performed RNA immunoprecipitation (RIP)-sequencing of AtGRP7-GFP plants subjected to formaldehyde fixation. 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. 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. 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. Functions for RNA-binding proteins in orchestrating plant development and environmental responses are well established.

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