Regulation of miRNA biogenesis in Arabidopsis - An RNA-affinity based approach to characterize pri-miRNA stem-loop binding proteins

拟南芥中 miRNA 生物合成的调控 - 一种基于 RNA 亲和力的方法来表征 pri-miRNA 茎环结合蛋白

• 批准号: 314773174
• 负责人: Dr. Tino Köster
• 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
• 财政年份: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2020-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/314773174?language=en
• 关键词: Regulation; miRNA; biogenesis; Arabidopsis; RNA

Regulation at the RNA level by RNA-binding proteins (RBPs) and small regulatory RNAs is key to coordinating eukaryotic gene expression. Among small RNAs are microRNAs (miRNAs) that are ~21nt-long single-stranded RNAs and regulate the expression of cognate target mRNAs by cleavage or inhibition of translation. The importance of miRNAs is highlighted by their regulatory functions in development, differentiation and environmental responses. MiRNAs are generated from double-stranded primary (pri)-miRNAs with internal stem-loop structures that are converted to short-lived stem-loop precursor (pre)-miRNAs and further processed to mature miRNAs. In contrast to their mammalian counterparts, stem-loops of plant miRNA precursors are diverse and highly structured which constitutes a basis for extensive regulation of miRNA biogenesis through direct binding of RBPs. However, trans-acting regulators of the biogenesis of specific miRNAs are largely unknown. Our goal is a comprehensive identification of proteins that bind to the stem-loops of pri-miRNAs and affect miRNA biogenesis in Arabidopsis thaliana. We will exploit an RNA-centric approach to pull down proteins directly binding to the stem-loops of selected miRNA precursors in vitro. Therefore, in vitro transcripts of selected pri-miRNA stem-loops will be tagged with the short recognition sequence of the CRISPR endoribonuclease Csy4. An enzymatically inactive variant, Csy4*, will be expressed in E.coli and affinity-purified. After biotinylation, Csy4* will be immobilized on streptavidin beads and the Csy4* beads will be loaded with the pri-miRNA stem-loops tagged with the Csy4 recognition sequence. The Csy4*-RNA beads will then be incubated with protein extracts prepared from Arabidopsis nuclei to allow binding of RBPs to the pri-miRNA stem-loops. Upon reactivation of the Csy4* nuclease activity by imidazole the Csy4* recognition sequence is cleaved off the pri-miRNA stem-loops and the RNA-protein complexes are released. The method for this affinity enrichment will be optimized based on the specific enrichment of the known trans-acting regulators of miRNA biogenesis and general processing factors binding to pri-miR398b. Subsequently, the procedure will be scaled up and co-precipitating proteins will be identified via mass spectrometry. Proteins which are significantly enriched with tagged pri-miRNAs loaded onto the Cys4* beads but not by beads carrying only the tag are candidates regulating pri-miRNA processing. Their physiological relevance in miRNA biogenesis will be analyzed in loss-of-function mutants. To obtain genome-wide insights into the extent and molecular mechanism of RBP action, direct in vivo targets and RBP binding sites will be identified.Overall, the project will deliver new insight into molecular mechanisms underlying posttranscriptional regulation of miRNA processing by plant RBPs and will ultimately advance our understanding of regulatory principles of eukaryotic miRNA expression.

RNA结合蛋白（RBP）和小的调节RNA在RNA水平上的调节是协调真核基因表达的关键。在小RNA中有微小RNA（miRNAs），其是~ 21 nt长的单链RNA，并且通过切割或抑制翻译来调节同源靶mRNA的表达。miRNAs在发育、分化和环境响应中的调节功能突出了其重要性。miRNA由具有内部茎环结构的双链初级（pri）-miRNA产生，所述双链初级（pri）-miRNA转化为短寿命茎环前体（pre）-miRNA并进一步加工为成熟miRNA。与它们的哺乳动物对应物相比，植物miRNA前体的茎环是多样的并且高度结构化的，这构成了通过直接结合RBP来广泛调节miRNA生物合成的基础。然而，特定miRNA的生物发生的反式作用调节剂在很大程度上是未知的。我们的目标是全面鉴定与pri-miRNA茎环结合并影响拟南芥中miRNA生物合成的蛋白质。我们将利用一种以RNA为中心的方法，在体外拉下与选定的miRNA前体的茎环直接结合的蛋白质。因此，所选pri-miRNA茎环的体外转录物将用CRISPR核糖核酸内切酶Csy 4的短识别序列标记。将在大肠杆菌中表达无酶活性的变体Csy 4 * 并亲和纯化。在生物素化后，Csy 4 * 将固定在链霉亲和素珠上，并且Csy 4 * 珠将装载有用Csy 4识别序列标记的pri-miRNA茎环。然后将Csy 4 *-RNA珠与从拟南芥核制备的蛋白质提取物一起孵育，以允许RBP与pri-miRNA茎环结合。在通过咪唑重新激活Csy 4 * 核酸酶活性时，Csy 4 * 识别序列从pri-miRNA茎环上切割下来，并释放RNA-蛋白质复合物。将基于miRNA生物发生的已知反式作用调节剂和结合pri-miR 398 b的一般加工因子的特异性富集来优化用于该亲和富集的方法。随后，该程序将扩大规模，并通过质谱法鉴定共沉淀蛋白质。显著富集有加载到Cys 4 * 珠上的标记的pri-miRNA但不富集仅携带标签的珠的蛋白质是调节pri-miRNA加工的候选物。将在功能丧失突变体中分析它们在miRNA生物发生中的生理相关性。为了获得全基因组范围的RBP作用的程度和分子机制的见解，直接在体内靶点和RBP的结合位点将被确定。总体而言，该项目将提供新的见解的转录后调控的miRNA加工的植物RBP的分子机制，并最终将推进我们对真核生物的miRNA表达的调控原则的理解。