在真核生物中，microRNA (miRNA) 是一类21至24个核苷酸 (nucleotide, nt) 的小RNA (small RNA, sRNA)，它们可在转录后水平通过切割靶标mRNA或抑制翻译调控基因表达。在植物中，RNase III家族蛋白Dicer-like 1 (DCL1)、双链RNA结合蛋白HYPONASTIC LEAVES 1 (HYL1) 和锌指蛋白Serrate (SE) 组成切割复合物，对初级miRNA (pri-miRNA) 进行加工，最终产生miRNA。切割复合物定位于细胞核中并形成2-4个直径为0.2-0.8 μm的切割小体 (dicing body, D-body)。我们前期研究表明，D-body是miRNA产生场所，由其核心组分SE通过液-液相分离 (liquid-liquid phase separation, LLPS) 驱动D-body形成。本论文研究了D-body的组分SICKLE (SIC) 和CYP71 (Cyclophilin-71) 对D-body组装和miRNA加工调控机制。SICKLE (SIC) 是一个富含脯氨酸具有高度无序氨基酸序列的蛋白，有液-液相分离潜力。本研究通过体内和体外实验证明，SIC具有液-液相分离属性。验证发现，SIC与D-body核心组分DCL1、HYL1、SE及pri-miRNA相互作用，并参与D-body组装。通过遗传和生化实验表明，SIC提高了DCL1切割反应效率，促进miRNA的产生。此外，本研究建立了DCL1体外切割的酶动力学参数测定方法，揭示了SIC通过提高DCL1反应Kcat促进了miRNA产生的动力学机制。最终研究表明，SIC通过相分离增强了与SE的相互作用，从而被高效招募富集在D-body中，促进miRNA的加工产生。另外，本研究利用SE可招募miRNA加工因子进入D-body这一活性，质谱鉴定到D-body新组分CYP71，同时本课题组前期通过针对miRNA通路遗传筛选，也获得了cyp71突变体。cyp71突变体中miRNA累积减少，miRNA靶标基因表达水平升高。显微成像观察表明，CYP71定位于D-body，参与D-body组装。CYP71是一个脯氨酰顺反异构酶，可以与SE直接相互作用，并通过其脯氨酰顺反异构活性增强SE相分离，促进D-body的组装形成。根据遗传和生化结果，CYP71促进miRNA产生依赖其脯氨酰顺反异构活性。综上，液-液相分离在植物miRNA产生过程中发挥着重要作用，本论文通过对拟南芥中SIC和CYP71参与D-body组装和调控产生miRNA的机制展开研究，阐明了D-body中液-液相分离对酶促反应的调控机制，从而更深入地理解了液-液相分离在生物大分子聚集体中的生物学意义。
In eukaryotes, microRNAs （miRNAs） are a class of 21-24 nucleotides （nt） small RNAs （small RNAs, sRNAs） that regulate gene expression at the post-transcriptional level by cleaving target mRNAs or repressing translation. In plants, the RNase III family protein Dicer-like 1 （DCL1）, the double-stranded RNA-binding protein HYPONASTIC LEAVES 1 （HYL1）, and the zinc finger protein Serrate （SE） form dicing complex that processes primary miRNAs （pri-miRNAs） into mature miRNAs. Dicing complexes are localized in the nucleus and forms 2-4 dicing bodies （D-bodies） of 0.2-0.8 μm in diameter. We have previously showed that the D-body is the site of miRNA processing. Liquid-liquid phase separation （LLPS） of SE drives dicing body assembly and promotes miRNA processing in Arabidopsis.To further investigate the mechanism of D-body assembly and regulation of miRNA processing, In this study, we identified and investigated the mechanisms of D-body components, SICKLE （SIC） and CYP71 （Cyclophilin-71）. SICKLE （SIC） is a proline-rich protein with a highly disordered amino acid sequence and has LLPS potential. In vivo and in vitro experiments demonstrated that SIC has LLPS properties. We verified that SIC interacts with D-body core components DCL1, HYL1, SE and pri-miRNA and participates in D-body assembly. Both genetic and biochemical experiments showed that SIC improved the efficiency of DCL1 cleavage reaction and promoted miRNA processing. In addition, we established an enzymatic kinetic parameters assay for DCL1 cleavage in vitro and revealed the kinetic mechanism by which SIC promotes miRNA production by increasing the DCL1 cleavage turn-over rate. We concluded that SIC enhanced the interaction with SE through phase separation, and thus was efficiently recruited to be enriched in D-body to promote miRNA processing.In Addition, SE can recruit miRNA processing factors into the D-body through LLPS, we identified a new D-body component CYP71 by mass spectrometry. We have also previously identified cyp71 mutants by genetic screening for miRNA pathway. cyp71 mutants showed reduced miRNA accumulation and increased expression levels of miRNA target genes. CYP71 is localized in D-body and is involved in D-body assembly. CYP71 can interact directly with SE and enhance SE phase separation through its prolyl cis-trans isomerization activity, to facilitate the formation of D-body assembly. Based on genetic and biochemical results, prolyl cis-trans isomerase activity of CYP71 is required for miRNA processing.In summary, LLPS plays an important role in the process of miRNA production in plants. In this thesis, we investigated the mechanism of SIC and CYP71 involved in D-body assembly and regulation of miRNA processing in Arabidopsis, and elucidates the mechanism of the enzymatic reaction regulation by liquid-liquid phase separation in D-body, which leads to a further insight of liquid-liquid phase separation in biomolecular condensates.
