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

RNA结合蛋白（rbp）和小调控RNA在RNA水平上的调控是协调真核生物基因表达的关键。小rna中有microRNAs (miRNAs)，它们是~21nt长的单链rna，通过切割或抑制翻译来调节同源靶mrna的表达。mirna在发育、分化和环境反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，这些mirna转化为短寿命的茎环前体(pre)- mirna，并进一步加工成成熟的mirna。与哺乳动物相比，植物miRNA前体的茎环具有多样性和高度结构化，这为通过直接结合rbp广泛调控miRNA的生物发生奠定了基础。然而，特定mirna生物发生的反式调节因子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miRNA生物发生的蛋白质。我们将利用以rna为中心的方法，在体外拉下直接与选定的miRNA前体的茎环结合的蛋白质。因此，选择的pri-miRNA茎环的体外转录本将被CRISPR核糖核酸内切酶Csy4的短识别序列标记。酶失活的变体Csy4*将在大肠杆菌中表达并进行亲和纯化。生物素化后，Csy4*将固定在链亲和素珠上，Csy4*珠将装载带有Csy4识别序列标记的pri-miRNA茎环。然后将Csy4*-RNA珠与拟南芥核制备的蛋白质提取物一起孵育，使rbp与pri-miRNA茎环结合。当Csy4*核酸酶活性被咪唑重新激活时，Csy4*识别序列从pri-miRNA茎环上被切割，rna -蛋白复合物被释放。基于已知的miRNA生物发生的反式作用调节因子和与pri-miR398b结合的一般加工因子的特异性富集，将优化这种亲和力富集的方法。随后，该程序将扩大规模，并通过质谱法鉴定共沉淀蛋白质。被标记的pri-miRNA显著富集的蛋白装载到Cys4*珠上，而不是只携带标签的珠上，这些蛋白是调节pri-miRNA加工的候选蛋白。它们在miRNA生物发生中的生理相关性将在功能缺失突变体中进行分析。为了在全基因组范围内深入了解RBP作用的程度和分子机制，将确定直接的体内靶点和RBP结合位点。总的来说，该项目将为植物rbp转录后调控miRNA加工的分子机制提供新的见解，并最终促进我们对真核miRNA表达调控原理的理解。

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.