However, up to 80% of the cDNAs terminate at the crosslinked nucleotide. Thus, CLIP can only identify sequences with read through of the RT beyond the crosslink site. For CLIP, adapters are attached to both the 5′ and 3′ ends of the RNAs co-precipitating with the protein of interest. To overcome this drawback, more recently developed crosslinking and immunoprecipitation (CLIP) techniques rely on UV-induced covalent bonds between RBPs and their target RNAs, providing information on the site of interaction. While RIP is useful to identify in vivo target transcripts, it does not provide immediate information about the binding motifs on the RNAs. In a first RIP-seq analysis in Arabidopsis, more than 4000 targets of the serine/arginine rich (SR)-like protein SR45 were identified by RNA immunoprecipitation, followed by high-throughput sequencing. RIP and subsequent identification of bound transcripts by reverse transcription (RT)-PCR has been used to confirm candidate in vivo targets of plant RBPs. In conventional RNA immunoprecipitation (RIP) techniques, formaldehyde is used for crosslinking. To preserve the physiological RNA–protein interactions, RNA and bound proteins are often crosslinked in vivo. In higher plants, RBPs were immunoprecipitated from lysates of purified maize chloroplasts under native conditions and RNAs were identified by microarrays. To date, global mapping of in vivo RNA–protein interactions is performed by immunopurification of RNA-binding proteins using antibodies against the native protein or an epitope, and cataloguing the associated RNAs by RNA-seq. The complete binding repertoire of any of these RBPs is virtually unknown. Arabidopsis thaliana harbors 197 proteins with an RNA recognition motif (RRM), the most frequent type of RNA-binding domain. This regulation at the RNA level represents an important checkpoint to extensively modulate gene expression once transcription has been initiated. RNA-binding proteins (RBPs) regulate RNA processing steps from synthesis to decay, including pre-mRNA splicing, transport, 3′ end formation, translation, and degradation. 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. Alternative splicing of AtGRP7 targets at LL24 vs. Changes in alternative splicing among AtGRP7 targets. Differentially expressed AtGRP7 targets at LL24. High-confidence binders identified by both iCLIP and RIP-seq at LL24. Preprocessing of the iCLIP sequencing libraries at LL24 and mapping statistics. Differentially expressed AtGRP7 targets at L元6. Pentamers enriched in the vicinity of the crosslink sites at L元6. High-confidence binders identified by both iCLIP and RIP-seq at L元6. (PDF 4700 kb)Īdditional file 2: Table S1: Preprocessing of the iCLIP sequencing libraries at L元6 and mapping statistics. Figure S15 Validation of differential alternative splicing of AtGRP7 targets at LL24. Validation of iCLIP or RIP-seq alternative splicing targets by RIP RT-PCR. Validation of differential alternative splicing of AtGRP7 targets at L元6. Changes in distribution of the log 2 fold changes of genes differentially expressed at LL24 (DEGs) in the grp7-1 8i mutant or AtGRP7-ox plants upon binding to AtGRP7. AtGRP7 targets differentially expressed at LL24 in plants with altered AtGRP7 levels. Position of the AtGRP7 crosslink sites within the transcripts at LL24. Differentially expressed AtGRP7 targets (DEGs) in plants with altered AtGRP7 level at L元6. iCLIP crosslink sites in the AtGRP7 transcript in comparison to in vitro binding sites. Clustering of motifs identified by MEME analysis. Scatterplot of RIP-seq data versus RNA-seq. iCLIP crosslink sites on AtGRP7 target transcripts. Significant crosslink sites in AtGRP7::GFP (GFP), AtGRP7::AtGRP7 R 49 Q - GFP (RQ), and AtGRP7::AtGRP7- GFP samples. Additional file 1: Figure S1: Monitoring for UV stress upon UV crosslinking.
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