Splicing Factors and riboregulators in the control of thermo-resilience in plants

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Global warming with increased frequencies of drought and temperature extremes affects agricul-ture. This process affects gene expression, and alternative pre-mRNA splicing (AS) contributes to adjust the plant transcriptome to temperature changes and plays a role in thermo-resilience. AS is exquisitely controlled by splicing factors that interact with cis-regulatory motifs in pre-mRNAs. More recently, the Arabidopsis long non-coding RNA (lncRNA) ALTERNATIVE SPLICING COMPETITOR (ASCO) has been identified as a novel regulator of AS. ASCO binds to the splicing fac-tor NUCLEAR SPECKLE RNA-BINDING PROTEIN (NSR) and competes with its splicing mRNA targets. Furthermore, a genome wide compendium of in vivo RNA targets has been established for the splicing factor GLYCINE-RICH RNA-BINDING PROTEIN 7 (GRP7), and both pre-mRNAs and lncRNAs have been identified, further supporting the idea that lncRNAs generally intervene in AS regulation.In RIBORES (Riboregulators in Thermo-Resilience), we will dissect the mechanism of lncRNA-mediated regulation of AS in order to develop a synthetic biology approach to manipulate AS in planta, using synthetic lncRNAs. Specifically, we will determine the mRNA and lncRNA tar-gets of NSR using individual nucleotide resolution crosslinking and immunoprecipitation (iCLIP) and delineate the specific NSR binding motifs in these RNAs. In a mutational analysis, we will dissect the sequence and structure requirements for ASCO function. In parallel, we will identify additional lncRNA targets of GRP7 different from ASCO. As proof-of-principle, through domain swapping, we will replace NSR binding sites on ASCO by GRP7 binding sites, and reciprocally, replace GRP7 binding sites on its lncRNA targets by NSR binding sites from ASCO. These con-structs will allow us to test whether we can manipulate the regulatory function of these splicing factors using synthetic lncRNAs. We will then assess the biological relevance of these synthetic lncRNAs in the riboregulation of thermoresilience, as both ASCO de-regulated and grp7 mutant plants are impaired in temperature responses. As GRP7 and NSR are highly conserved in plants, we will translate this approach to toma-to plants, which are heat sensitive. We already have identified the impact of heat stress on AS in tomato and have investigated the heat response of the NSR and GRP7 orthologues. The intro-duction of synthetic lncRNAs as well as the identification of a set of heat responsive lncRNAs in tomato will support the notion that lncRNA-mediated regulation of AS is a general mechanism in plants. Overall, RIBORES may provide new RNA-based strategies to regulate AS programs in eukaryotes, opening broad perspectives to develop innovative biotechnological tools to modulate gene expression in the context of agriculture and health.