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

全球变暖，干旱和极端温度的频率增加，影响了农业。这一过程影响了基因的表达，而选择性pre-mRNA剪接（AS）有助于调节植物转录组以适应温度变化，并在抗热性中发挥作用。AS是由剪接因子精细控制的，剪接因子与pre- mrna中的顺式调控基序相互作用。最近，拟南芥长链非编码RNA （lncRNA）选择性剪接竞争者（ASCO）被发现是一种新的as调节因子。ASCO结合剪接因子核斑rna结合蛋白（NUCLEAR SPECKLE rna binding PROTEIN， NSR），并与其剪接mRNA靶标竞争。此外，针对剪接因子GLYCINE-RICH RNA- binding PROTEIN 7 （GRP7）建立了一个全基因组范围的体内RNA靶点纲要，并鉴定出了pre- mrna和lncRNAs，进一步支持了lncRNAs通常干预AS调控的观点。在RIBORES（热弹性中的核糖调节因子）中，我们将剖析lncrna介导的AS调节机制，以便开发一种利用合成lncrna操纵植物AS的合成生物学方法。具体来说，我们将使用单个核苷酸分辨率交联和免疫沉淀（iCLIP）来确定NSR的mRNA和lncRNA靶标，并描述这些rna中特定的NSR结合基序。在突变分析中，我们将剖析ASCO功能的序列和结构要求。同时，我们将确定与ASCO不同的GRP7的其他lncRNA靶点。作为原理证明，我们将通过域交换将ASCO上的NSR结合位点替换为GRP7结合位点，反过来，将其lncRNA靶标上的GRP7结合位点替换为ASCO上的NSR结合位点。这些结构将允许我们测试我们是否可以使用合成lncrna操纵这些剪接因子的调节功能。然后，我们将评估这些合成lncrna在热恢复性核糖调节中的生物学相关性，因为ASCO去调控和grp7突变植物在温度反应中都受损。由于GRP7和NSR在植物中高度保守，我们将把这种方法应用于热敏植物番茄。我们已经确定了热胁迫对番茄AS的影响，并研究了NSR和GRP7同源物的热响应。合成lncrna的引入以及在番茄中发现的一组热响应lncrna将支持lncrna介导的as调控是植物中普遍机制的观点。总的来说，RIBORES可能为真核生物调控AS程序提供新的基于rna的策略，为开发创新的生物技术工具来调节农业和健康背景下的基因表达开辟了广阔的前景。

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.