At-RS31 orchestrates hierarchical cross-regulation of splicing factors and integrates alternative splicing with TOR-ABA pathways

Abstract

Alternative splicing is essential for plants, enabling a single gene to produce multiple transcript variants to boost functional diversity and fine-tune responses to environmental and developmental cues. At-RS31, a plant-specific splicing factor in the Serine/Arginine (SR)-rich protein family, responds to light and the Target of Rapamycin (TOR) signaling pathway, yet its downstream targets and regulatory impact remain unknown.To identify At-RS31 targets, we applied individual-nucleotide resolution crosslinking and immunoprecipitation (iCLIP) and RNAcompete assays. Transcriptomic analyses of At-RS31 mutant and overexpressing plants further revealed its effects on alternative splicing.iCLIP identified 4,034 At-RS31 binding sites across 1,421 genes, enriched in CU-rich and CAGA RNA motifs. Comparative iCLIP and RNAcompete data indicate that the RS domain of At-RS31 may influence its binding specificity in planta, underscoring the value of combining in vivo and in vitro approaches. Transcriptomic analysis showed that At-RS31 modulates diverse splicing events, particularly intron retention and exitron splicing, and influences other splicing modulators, acting as a hierarchical regulator.By regulating stress-response genes and genes in both TOR and abscisic acid (ABA) signaling pathways, At-RS31 may help integrate these signals, balancing plant growth with environmental adaptability through alternative splicing.

Keywords: ABA; Arabidopsis; RNAcompete; SR proteins; TOR kinase; alternative splicing; binding site; iCLIP.

Fig. 1

Determination of At-RS31 in vivo binding sites by iCLIP (a) Left: Autoradiogram of RS31-GFP and GFP protein-RNA complexes. After UV crosslinking, cell lysates were subjected to immunoprecipitation with GFP Trap beads. RNAs were radioactively labelled, and the complexes were separated by denaturing gel electrophoresis. IN, input (lysate). Treatment of the precipitate with RNase I (+ RNase) indicates the size of the precipitated proteins. The region above the fusion protein containing the co-precipitated RNAs used for library preparation is indicated. Right: iCLIP western blot. Immunoblot analysis of the membrane shown in the left panel with anti-GFP antibody. Bands for GFP and RS31-GFP are marked accordingly. (b) Distribution of the At-RS31 binding sites within protein-coding transcripts. Distribution of RS31-GFP and GFP binding sites within transcripts in relation to the total length of the transcript features. 5’ UTR and 3’ UTR - 5’ and 3’ untranslated regions; CDS - coding sequences. (c) Distribution of the At-RS31 binding sites within 5’ UTRs relative to the transcription start sites (TSS). Distance to TSS is shown in nucleotides (nt). (d) Distribution of At-RS31 binding sites peaking upstream of 5’ splice sites (5’SS). Only exons at least 50 nucleotides in length were analyzed. The red line represents the local density of binding sites. The blue box marks the −35-20 nt region upstream of 5’SS where RS31-GFP binding sites are enriched. (e) Significant STREME binding site motifs. Sequence logos of the most significant (based on their p-value) RS31-GFP binding motifs identified by STREME analysis. For the analysis, only sequences from the 9-nucleotide binding site regions were considered. (f) Hexamer counts and z-scores. Scatterplot showing hexamer frequencies and counts computed from the 9-nucleotide binding site sequences of the RS31-GFP iCLIP sample. Hexamer counts (x-axis) are compared against hexamer z-scores (y-axis). The most enriched hexamers and the ones with highest counts are labelled according to their sequence. The highlighted hexamers (red) contain the subsequence CAGA. (g) iCLIP binding site distance to RNAcompete motif sites. Distribution of RS31-GFP binding site peaks in relation to CAGA sites across At-RS31 target transcripts. The position 1 on the x-axis denotes to the C in the CAGA motif identified by the RNAcompete assay.

Fig. 2

Identification of At-RS31 RNA-binding motifs using RNAcompete (a) Overview of the RNAcompete assay. GST-tagged full-length At-RS31 protein and its truncated version comprising both RRMs were incubated with a 75-fold molar excess of designed RNA pool. RNA bound to GST-RS31 and GST-RS31RRMs fusion proteins during the GST pulldown was eluted, purified, labeled and hybridized to custom Agilent 244K microarrays. Microarray data was analysed computationally to identify 7-mers specifically bound by At-RS31 and generate RNA-binding motifs. RRM1 and RRM2 – RNA recognition motif domains; RS – region rich in arginines and serines. (b) RNA-binding motifs of the full-length At-RS31 and its truncated version containing RRMs identified in the RNAcompete assay and represented as sequence logos.

Fig. 3

At-RS31 and its impact on alternative splicing and gene expression in Arabidopsis thaliana (a) Schematic of the At-RS31 protein domain structure. The protein contains two RNA recognition motifs (RRM1 and RRM2) and an arginine/serine-rich region (RS). (b) Structure of At-RS31 gene, its alternative splicing events and transcript models. Carets indicate alternative splicing events in the second intron. The solid caret represents the splicing event that produces the reference transcript AT3G61860.1 (mRNA1), which encodes the At-RS31 protein. Retention of the second intron generates the mRNA4 transcript. The dashed caret marks the use of an alternative 3’ splice site, resulting in the mRNA3 transcript. Dotted carets denote an alternative exon generated via use of both alternative 3’ and 5’ splice sites, producing the mRNA2 variant. PTC - the premature termination codon. Regions from the AUG start codon to the reference stop codon (mRNA1) or the PTC (mRNA2-4) are shaded in black. The arrowhead indicates the T-DNA insertion site in the SALK_021332 line (rs31-1 mutant). Black arrows show positions of primers used for RT-PCR analysis in (C). (c) RT-PCR analysis of At-RS31 transcript levels in the A. thaliana wild type, 35S::RS31 (overexpression of the mRNA1 under the CaMV 35S promoter), and rs31-1 plants. Ubiquitin (UBQ) was used as a loading control. (d) Phenotypes of wild type, 35S::RS31 overexpression, and rs31-1 mutant plants used for RNA-sequencing. Plants were grown under a 16 h light/8 h dark cycle at 22°C on vertical agar plates containing half-strength MS medium. (e) Distribution of alternative splicing event types differentially regulated in rs31-1 and 35S::RS31 lines in comparison to wild type A. thaliana. Diagrams on the left illustrate the analyzed alternative splicing event types: AA (alternative acceptor, or alternative 3’ splice site), AD (alternative donor, or alternative 5’ splice site), CE (cassette exon), EI (exitron), and RI (retained intron). (f) Proportions of differential exitron (EI) and retained intron (RI) events with reduced or increased splicing efficiency in rs31-1 and 35S::RS31 lines compared to wild type. Positive and negative percentages indicate the proportion of events with increased or decreased Percent Spliced In values (ΔPSI), respectively. (g) Comparison of differential alternative splicing (DAS) and iCLIP statistics. Venn diagram shows the overlap between genes with At-RS31 iCLIP binding sites and DAS genes in rs31-1 and 35S::RS31 lines compared to wild type. (h) Comparison of DAS and DEG statistics. Venn diagram shows numbers of genes differentially alternatively spliced (DAS) and differentially expressed (DEG) in rs31-1 and 35S::RS31 lines compared to wild type.

Fig. 4.

Regulation of Arabidopsis SR protein family by At-RS31 (a) Overview of differential alternative splicing (DAS) and differential gene expression (DE) in Arabidopsis SR genes in rs31-1 mutant and 35S::RS31 overexpression plants, compared to wild-type controls. The table includes information on iCLIP peaks and the top 7-mers identified by RNAcompete within SR genes. “NA” indicates data not applicable. (b) Integrated Genomics Viewer (IGV) tracks depicting iCLIP cross-link sites for GFP and RS31-GFP, along with RNA-seq read coverage tracks for rs31-1, 35S::RS31, and wild-type plants. Transcript models for SR genes are displayed using the Boxify tool (https://boxify.boku.ac.at/). Protein-coding regions, spanning from the translational start codon to the stop codon or premature termination codon, are shown in black. Red vertical lines represent the locations of At-RS31 binding sites identified by iCLIP. Only transcripts relevant to the identified DAS events are displayed, with dashed rectangles marking these alternatively spliced regions. A black line denotes the position of an upstream open reading frame (uORF) in At-SR34a. Black upward and downward arrows indicate an increase or decrease, respectively, in the ratio of splice variants encoding full-length SR proteins.

Fig. 5.

At-RS31 is involved in regulating abscisic acid pathway (a) Phenotype of At-RS31 mutant and overexpression plants in response to abscisic acid. Seeds of rs31-1 mutant, 35S::RS31 overexpression, and wild-type plants were germinated and grown on the vertical MS agar plates supplemented with 0, 0.5, 1, and 2 μM of abscisic acid at 23°C, 16h light/8h dark. (b) At-RS31 modulates alternative splicing of HAB1 (Hypersensitive to ABA1). Integrated Genomics Viewer (IGV) tracks show iCLIP cross-link sites for GFP and RS31-GFP, along with RNA-seq read coverage for rs31-1, 35S::RS31, and wild-type plants. In the transcript models, protein-coding regions, spanning from the translational start codon to the stop codon or premature termination codon, are shown in black. Only transcripts relevant to the identified DAS events are displayed, with dashed rectangles marking these alternatively spliced regions.