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Gene regulation dependent on N6-methyladenosine (m6A) in mRNA involves RNA-binding proteins that recognize m6A through a YTH domain. The Arabidopsis YTH-domain protein ECT2 is thought to influence mRNA 3′-end formation via binding to URU(m6A)Y sites, an unexpected conclusion given that ECT2 functions require its m6A binding activity, and that RR(m6A)CH is the m6A consensus site in all eukaryotes. Here, we apply the orthogonal techniques individual nucleotide-resolution UV-crosslinking and immunoprecipitation (iCLIP) and HyperTRIBE to define high-quality target sets of the YTH-domain proteins ECT2 and ECT3. The results show that in vivo, ECT2 does in fact bind to RR(m6A)CH. URUAY and other pyrimidine-rich motifs are enriched around, but not at m6A-sites, reflecting a preference for N6-adenosine methylation of RRACH islands in pyrimidine-rich regions. Such regions may also be implicated in ECT2-binding. In particular, a series of properties unique to the URUAY motif suggest that URUAY-type sequences act as sites of competition between unknown RNA-binding proteins and the intrinsically disordered region of ECT2. We also show that the abundance of many ECT2/3 mRNA targets is decreased in meristematic cells devoid of ECT2/3/4-activity. In contrast, loss of ECT2/3/4 activity has no effect on polyadenylation site usage in ECT2/3 targets, consistent with the exclusive cytoplasmic localization of ECT2 observed by super-resolution confocal microscopy. Our study reconciles conflicting results between genetic observations on N6-adenosine methylation and ECT2/3/4 function on the one side, and ECT2 target identification on the other, and point to regulation of cytoplasmic mRNA function, including abundance, as a mechanism of plant YTHDF action.

The RNA-binding proteome plays a key role in controlling every step in the life of RNA molecules. Through interaction with dedicated sequence motifs, RNA-binding proteins coordinate processing of cohorts of genes. Understanding such posttranscriptional networks controlled by an RNA-binding protein requires a comprehensive identification of its in vivo targets. In Arabidopsis thaliana, RNA immunoprecipitation followed by reverse transcription-PCR has been widely used to test the association of candidate targets with RNA-binding proteins. The detection of unknown target transcripts requires methods operating at the level of the entire transcriptome. Here, we describe a protocol for RNA immunoprecipitation coupled to the generation of libraries from the co-purified RNAs for high-throughput sequencing. This allows determining RNAs associated with RNA-binding proteins in planta at a global scale.

Additional file 2 Installation and usage instructions. SEQing installation instructions and command line options with use cases and descriptions.

Additional file 3 supplemental figures containing screenshots of the details tab, settings tab and a visualization example of a public human dataset (GSE99700). Screenshots of SEQing’s Details tab to deliver additional information on the selected gene to the browsing user (Supplemental Figure 1) and customization options in the Settings tab (Supplemental Figure 2). The screenshots were taken using the sample data incorporated in the github repository. Supplemental Figure 3 shows crosslink sites from a public human dataset (GSE99700) visualized using SEQing.

Monitoring for UV stress upon UV crosslinking. Related to Fig. 1. Figure S2. iCLIP of AtGRP7-GFP. Figure S3. Significant crosslink sites in AtGRP7::GFP (GFP), AtGRP7::AtGRP7 R 49 Q -GFP (RQ), and AtGRP7::AtGRP7-GFP samples. Figure S4. iCLIP crosslink sites on AtGRP7 target transcripts. Related to Fig. 3. Figure S5. Scatterplot of RIP-seq data versus RNA-seq. Figure S6. Clustering of motifs identified by MEME analysis. Related to Fig. 5. Figure S7. iCLIP crosslink sites in the AtGRP7 transcript in comparison to in vitro binding sites. Figure S8. Differentially expressed AtGRP7 targets (DEGs) in plants with altered AtGRP7 level at LL36. Figure S9. Position of the AtGRP7 crosslink sites within the transcripts at LL24. Related to Fig. 2. Figure S10. Candidate binding motifs LL24. Related to Fig. 5. Figure S11. AtGRP7 targets differentially expressed at LL24 in plants with altered AtGRP7 levels. Figure S12. Changes in distribution of the log2 fold changes of genes differentially expressed at LL24 (DEGs) in the grp7-1 8i mutant or AtGRP7-ox plants upon binding to AtGRP7. Related to Fig. 6. Figure S13. Validation of differential alternative splicing of AtGRP7 targets at LL36. Figure S14. Validation of iCLIP or RIP-seq alternative splicing targets by RIP RT-PCR. Figure S15 Validation of differential alternative splicing of AtGRP7 targets at LL24. (PDF 4700 kb)

Alternative pre-messenger RNA splicing in higher plants emerges as an important layer of regulation upon exposure to exogenous and endogenous cues. Accordingly, mutants defective in RNA-binding proteins predicted to function in the splicing process show severe phenotypic alterations. Among those are developmental defects, impaired responses to pathogen threat or abiotic stress factors, and misregulation of the circadian timing system. A suite of splicing factors has been identified in the model plant Arabidopsis thaliana. Here we summarize recent insights on how defects in these splicing factors impair plant performance.

Plants overexpressing the RNA-binding protein AtGRP7 (AtGRP7-ox plants) constitutively express the PR-1 (PATHOGENESIS-RELATED-1), PR-2 and PR-5 transcripts associated with salicylic acid (SA)-mediated immunity and show enhanced resistance against Pseudomonas syringae pv. tomato (Pto) DC3000. Here, we investigated whether the function of AtGRP7 in plant immunity depends on SA. Endogenous SA was elevated fivefold in AtGRP7-ox plants. The elevated PR-1, PR-2 and PR-5 levels were eliminated upon expression of the salicylate hydroxylase nahG in AtGRP7-ox plants and elevated PR-1 levels were suppressed by sid (salicylic acid deficient) 2-1 that is impaired in SA biosynthesis. RNA immunoprecipitation showed that AtGRP7 does not bind the PR-1 transcript in vivo, whereas it binds PDF1.2. Constitutive or inducible AtGRP7 overexpression increases PR-1 promoter activity, indicating that AtGRP7 affects PR-1 transcription. In line with this, the effect of AtGRP7 on PR-1 is suppressed by npr (non-expressor of PR genes) 1. Whereas AtGRP7-ox plants restricted growth of Pto DC3000 compared with wild type (wt), sid2-1 AtGRP7-ox plants allowed more growth than AtGRP7-ox plants. Furthermore, we show an enhanced hypersensitive response triggered by avirulent Pto DC3000 (AvrRpt2) in AtGRP7-ox compared with wt. In sid2-1 AtGRP7-ox, an intermediate phenotype was observed. Thus, AtGRP7 has both SA-dependent and SA-independent effects on plant immunity. (2013 John Wiley & Sons Ltd.)