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

剪接因子和核糖调节剂在植物耐热性控制中的作用

• 批准号: 505658853
• 负责人: Professorin Dr. Dorothee Staiger
• 金额: --
• 依托单位: Arbeitsgruppe RNA Biologie und Molekulare Physiologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/505658853?language=en
• 关键词: Splicing; Factors; riboregulators; control; thermo

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.

全球变暖，干旱和极端温度的频率增加，影响了农业。这一过程影响基因的表达，而选择性的前mRNA剪接(AS)有助于调节植物转录组对温度变化的适应，并在耐热性中发挥作用。AS受到剪接因子的巧妙控制，这些剪接因子与前mRNAs中的顺式调控基序相互作用。最近，拟南芥长非编码RNA(LncRNA)选择性剪接竞争对手(ASCO)被确定为AS的一个新的调节因子。ASCO与剪接因子核斑点RNA结合蛋白(NSR)结合，并与其剪接mRNA靶标竞争。此外，富含甘氨酸的RNA结合蛋白7(GRP7)的体内RNA靶标已经建立在全基因组范围内，并且已经确定了前mRNAs和lncRNAs，进一步支持了lncRNAs通常参与AS调节的想法。在RIBORES(热弹性核调节因子)中，我们将剖析lncRNA介导的AS调节的机制，以便开发一种合成生物学方法来利用合成的lncRNAs来操纵植物中的AS。具体地说，我们将使用单个核苷酸分辨交联和免疫沉淀(ICLIP)来确定NSR的mRNA和lncRNA目标，并在这些RNA中描绘特定的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，我们将确定不同于ASCO的GRP7的额外LncRNA靶点。作为原则证明，我们将通过结构域交换，将ASCO上的NSR结合位点替换为GRP7结合位点，并反过来将其lncRNA靶标上的GRP7结合位点替换为ASCO的NSR结合位点。这些结构将使我们能够测试是否可以使用合成的lncRNA来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lncRNAs在热弹性核转录调节中的生物学相关性，因为ASCO去调节和grp7突变植物在温度反应中都受到损害。由于GRP7和NSR在植物中高度保守，我们将把这种方法转化到对热敏感的植物TOMA上。我们已经确定了热胁迫对番茄AS的影响，并研究了NSR和GRP7同源基因的热响应。人工合成的lncRNA的引入以及一组热响应lncRNA在番茄中的鉴定将支持这一观点，即lncRNA介导的AS调节是植物的一种普遍机制。总体而言，RIBORES可能提供新的基于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。