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

全球变暖，干旱和极端温度频率增加，影响农业。这一过程影响基因表达，而前体mRNA选择性剪接（AS）有助于调节植物转录组以适应温度变化，并在耐热性中发挥作用。AS由剪接因子精细控制，剪接因子与前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）选择性剪接竞争体（ASCO）已被鉴定为AS的新调节因子。ASCO与剪接因子核斑点RNA结合蛋白（NSR）结合，并与其剪接mRNA靶竞争。 此外，已经为剪接因子富含甘氨酸的RNA结合蛋白7（GRP 7）建立了体内RNA靶点的全基因组纲要，并且已经鉴定了前mRNA和lncRNA，进一步支持lncRNA通常干预AS调节的想法。（Riboregulators in Thermo-Resilience）我们将剖析lncRNA介导的AS调节机制，以便开发一种合成生物学方法，使用合成lncRNA在植物中操纵AS。具体而言，我们将使用单个核苷酸解析交联和免疫沉淀（iCLIP）来确定NSR的mRNA和lncRNA靶标，并描绘这些RNA中的特异性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，我们将鉴定不同于ASCO的GRP 7的其他lncRNA靶标。作为原理证明，通过结构域交换，我们将用GRP 7结合位点替换ASCO上的NSR结合位点，并且将其lncRNA靶上的GRP 7结合位点替换为来自ASCO的NSR结合位点。这些结构将使我们能够测试我们是否可以使用合成的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的lncRNA在热弹性的核糖调节中的生物相关性，因为ASCO去调节和grp 7突变体植物在温度响应中受损。由于GRP 7和NSR在植物中高度保守，我们将这种方法转化为对热敏感的番茄植物。我们已经确定了热应激对番茄AS的影响，并研究了NSR和GRP 7直系同源物的热反应。合成lncRNA的引入以及番茄中一系列热响应lncRNA的鉴定将支持lncRNA介导的AS调节是植物中的普遍机制的观点。总的来说，RIBORES可以提供新的基于RNA的策略来调节真核生物中的AS程序，为开发创新的生物技术工具以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.