Nuclear Speckle RNA Binding Proteins Remodel Alternative Splicing and the Non-coding Arabidopsis Transcriptome to Regulate a Cross-Talk Between Auxin and Immune Responses Jérémie Bazin, Natali Romero, Richard Rigo, Céline Charon, Thomas Blein, Federico Ariel, Martin Crespi To cite this version: Jérémie Bazin, Natali Romero, Richard Rigo, Céline Charon, Thomas Blein, et al.. Nuclear Speckle RNA Binding Proteins Remodel Alternative Splicing and the Non-coding Arabidopsis Transcriptome to Regulate a Cross-Talk Between Auxin and Immune Responses. Frontiers in Plant Science, Frontiers, 2018, 9, pp.1-13. 10.3389/fpls.2018.01209. hal-02379622 HAL Id: hal-02379622 https://hal.archives-ouvertes.fr/hal-02379622 Submitted on 26 May 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Distributed under a Creative Commons Attribution| 4.0 International License fpls-09-01209 August 21, 2018 Time: 12:34 # 1 ORIGINAL RESEARCH published: 21 August 2018 doi: 10.3389/fpls.2018.01209 Nuclear Speckle RNA Binding Proteins Remodel Alternative Splicing and the Non-coding Arabidopsis Transcriptome to Regulate a Cross-Talk Between Auxin and Immune Responses Jérémie Bazin1*, Natali Romero1, Richard Rigo1, Celine Charon1, Thomas Blein1, Federico Ariel1,2 and Martin Crespi1* 1 CNRS, INRA, Institute of Plant Sciences Paris-Saclay IPS2, Univ Paris Sud, Univ Evry, Univ Paris-Diderot, Sorbonne Paris-Cite, Universite Paris-Saclay, Orsay, France, 2 Instituto de Agrobiotecnologıa del Litoral, CONICET, Universidad Nacional del Litoral, Santa Fe, Argentina Nuclear speckle RNA binding proteins (NSRs) act as regulators of alternative splicing Edited by: Dorothee Staiger, (AS) and auxin-regulated developmental processes such as lateral root formation in Bielefeld University, Germany Arabidopsis thaliana. These proteins were shown to interact with specific alternatively Reviewed by: spliced mRNA targets and at least with one structured lncRNA, named Alternative Rossana Henriques, Splicing Competitor RNA. Here, we used genome-wide analysis of RNAseq to monitor University College Cork, Ireland Heike Lange, the NSR global role on multiple tiers of gene expression, including RNA processing UPR2357 Institut de Biologie and AS. NSRs affect AS of 100s of genes as well as the abundance of lncRNAs Moléculaire des Plantes (IBMP), CNRS, France particularly in response to auxin. Among them, the FPA floral regulator displayed *Correspondence: alternative polyadenylation and differential expression of antisense COOLAIR lncRNAs Jérémie Bazin in nsra/b mutants. This may explains the early flowering phenotype observed in nsra [email protected] and nsra/b mutants. GO enrichment analysis of affected lines revealed a novel link of Martin Crespi [email protected] NSRs with the immune response pathway. A RIP-seq approach on an NSRa fusion protein in mutant background identified that lncRNAs are privileged direct targets of Specialty section: NSRs in addition to specific AS mRNAs. The interplay of lncRNAs and AS mRNAs in This article was submitted to Plant Cell Biology, NSR-containing complexes may control the crosstalk between auxin and the immune a section of the journal response pathway. Frontiers in Plant Science Received: 16 May 2018 Keywords: RNA binding proteins, RNP complexes, alternative splicing, immune response, auxin Accepted: 27 July 2018 Published: 21 August 2018 Citation: INTRODUCTION Bazin J, Romero N, Rigo R, Charon C, Blein T, Ariel F and RNA binding proteins (RBPs) have been shown to affect all steps of post-transcriptional gene Crespi M (2018) Nuclear Speckle expression control, including alternative splicing (AS), silencing, RNA decay, and translational RNA Binding Proteins Remodel control (Bailey-Serres et al., 2009). The Arabidopsis thaliana genome encodes for more than 200 Alternative Splicing proteins predicted to bind RNAs. The picture becomes even more complex since over 500 proteins and the Non-coding Arabidopsis were found to bind polyAC RNA in a recent study attempting to define the RNA interactome using Transcriptome to Regulate affinity capture and proteomics (Marondedze et al., 2016). However, only a small subset of RBPs a Cross-Talk Between Auxin and Immune Responses. has been functionally assigned in plants. The versatility of RBPs on gene expression regulation has Front. Plant Sci. 9:1209. been recently highlighted by the identification of several among them acting at multiple steps of doi: 10.3389/fpls.2018.01209 post-transcriptional gene regulation (Lee and Kang, 2016; Oliveira et al., 2017). During mRNA Frontiers in Plant Science| www.frontiersin.org 1 August 2018| Volume 9| Article 1209 fpls-09-01209 August 21, 2018 Time: 12:34 # 2 Bazin et al. Genome-Wide Roles of NSR Proteins maturation, the transcript acquires a complex of proteins at each significantly up and down-regulated in nsra/b, compared exon–exon junction during pre-mRNA splicing that influences to wild type (Figure 1B and Supplementary Table S1B). the subsequent steps of mRNA translation and decay (Maquat, Principal component analysis (PCA) showed a dispersion of 2004). Although all RBPs bind RNA, they exhibit different RNA- the data compatible with statistical comparisons between groups sequence specificities and affinities. As a result, cells are able (Supplementary Figure S1). Multifactor analysis of differential to generate diverse ribonucleoprotein complexes (RNPs) whose gene expression further showed that nsra/b mutation has a major composition is unique to each mRNA and these complexes are effect on auxin-regulated gene expression. Indeed, a set of 951 further remodeled during the life of the mRNA in order to genes showed significant interaction between genotype and auxin determine its fate. One approach to determine RBP function regulation (Figure 1C and Supplementary Table S1B). This is consisted in the identification of all interacting molecules (the so- in agreement with our previous findings indicating that NSRs called RNPome) of a specific RNP and the conditions of their mediate auxin regulation of gene expression (Bardou et al., 2014). association. The ribonucleoprotein immunopurification assay We have previously shown that NSRs modulate auxin-induced facilitates the identification and quantitative comparison of RNA AS of a particular subset of genes using specific qRT-PCR assays association to specific proteins under different experimental (Bardou et al., 2014). We use now our RNA-seq dataset to conditions. This approach has been successfully used to elucidate characterize genome-wide effects of NSRs on AS and more the genome-wide role of a number of plant RBPs involved in generally on RNA processing (Figure 1A). To this end, we pre-mRNA splicing, stress granule formation or translational made use of the RNAprof software, which implements a control (Sorenson and Bailey-Serres, 2014; Gagliardi and gene-level normalization procedure and can compare RNA-seq Matarazzo, 2016; Foley et al., 2017; Köster and Meyer, 2018). read distributions on transcriptional units to detect significant The nuclear speckle RNA binding proteins (NSRs) are a profile differences. This approach allows de novo identification family of RBPs that act as regulators of AS and auxin regulated of RNA processing events independently of any gene feature or developmental processes such as lateral root formation in annotation independently of gene expression differences (Tran Arabidopsis thaliana. These proteins were shown to interact with et al., 2016). RNAprof results were parsed to retain only highly some of their alternatively spliced mRNA targets and at least with significant differential RNA processing events (p.adj < 10e-4) one structured lncRNA, named Alternative Splicing Competitor and further crossed with gene annotation in order to classify RNA (ASCO)(Bardou et al., 2014). Overexpression of ASCO was them according to their gene features. The majority of events shown to affect AS of a subset of mRNA regulated by NSRs, overlapped with intronic regions (Figure 1D and Supplementary similar to nsra/b double mutants, and ASCO was also shown to Table S1C), which is in accordance with data showing that intron compete in vitro with the binding of one AS mRNA target. This retention is the major event of AS in plants (Ner-Gaon et al., study suggested that plant lncRNAs are able to modulate AS of 2004). The effect of nsra/b on RNA processing and splicing is mRNA by hijacking RBPs, such as NSRs, involved in splicing enhanced in response to NAA. In other words the vast majority (Romero-Barrios et al., 2018). In addition, transcriptome analysis of differential events between nsra/b and wild type plants were using microarrays and specific AS analysis on a subset of mRNAs identified essentially in presence of auxin. suggested a role of NSR in transcriptome remodeling in response To further support the results from RNAprof and to gain to auxin (Bardou et al., 2014). knowledge on the functional consequences of NSR mediated Here we used genome wide analysis to monitor the NSR AS events, we quantified mRNA transcript isoforms of the global role on multiple