小RNA甲基转移酶HEN1在莱茵衣藻miRNA生物合成途径的作用机制研究

Methyltransferase HEN1 methylates the miRNA/miRNA* duplexes in the miRNA biosynthesis pathway, which helps maintain the stability of duplexes and helps prevent exonucleolytic degradation by uridylation. HEN1 has been extensively studied in higher multicellular organisms, however its biofunction and mechanism are not yet clear in unicellular eukaryotes at present. Our previous research showed Chlamydomonas HEN1 has a typical methyltransferase domain, which is very similar to the reported Arabidopsis AtHEN1. In addition, the miRNA expression is generally down-regulated in a Chlamydomonas loss-of-function mutant hen1. In this project, the miRNA methyltransferase activity of Chlamydomonas HEN1 will be verified in vitro. And the interaction between HEN1 and CrDCL3, DUS16 will be conducted using yeast two hybridization system. Moreover, the changes of the miRNA size, quantity and expression abundance of hen1 mutant will also be investigated, according to the miRNA sequencing analysis of hen1. Synergistically clarify the function and mechanism of Chlamydomonas HEN1 in the miRNA biosynthesis pathway, which are of great significance for revealing the origin and evolution of miRNA regulation system.

甲基转移酶HEN1在miRNA生物合成途径中通过甲基化修饰双链miRNA，保护其稳定而不被尿苷化降解。HEN1在高等多细胞生物中研究较多，而在低等单细胞生物中的具体功能及作用机制目前尚不清楚。申请者前期研究发现低等单细胞生物莱茵衣藻HEN1具有一个典型的甲基转移酶结构域，与已报道的高等植物拟南芥非常相似，还发现衣藻HEN1功能丧失型突变株hen1的miRNA表达普遍下调。本项目拟体外验证莱茵衣藻HEN1的甲基转移酶活性；利用酵母双杂交系统验证HEN1与miRNA合成途径重要蛋白CrDCL3、DUS16的相互作用；结合衣藻hen1突变体的miRNA测序分析，探知HEN1功能缺失后对衣藻miRNA大小、数量和表达丰度的影响，协同阐明HEN1在低等生物衣藻miRNA生物合成途径的功能及其作用机制。这对于揭示miRNA调控系统的起源和进化具有重要意义。

甲基转移酶HEN1在miRNA生物合成途径中通过甲基化修饰双链miRNA，保护其稳定而不被尿苷化降解。HEN1在高等多细胞生物中研究较多，而在低等单细胞生物中的具体功能及作用机制目前尚不清楚。前期研究生物信息学研究分析发现低等单细胞生物莱茵衣藻CrHEN1具有一个典型的甲基转移酶结构域，与已报道的高等植物拟南芥非常相似；此外CrHEN1还含有绿藻特有的功能结构域，核定位信号以及核输出信号。进化树分析发现CrHEN1与其它绿藻聚在一类，进化上比高等动植物早。miRNA组学分析发现莱茵衣藻CrHEN1功能丧失的突变株Crhen1中大量miRNA表达下调，进一步通过qRT-PCR和NorthernBlot分析验证了miRNA差异表达的真实性。通过体外β消除实验验证了莱茵衣藻CrHEN1具有甲基转移酶的活性。亚细胞定位实验表明CrHEN1主要定位于细胞核。体外ITC实验表明CrHEN1与DCL 3相互作用。功能互补实验表明CrHEN1不仅可回复衣藻Crhen1突变体表型，还可回复拟南芥Athen1突变体表型。由此说明CrHEN1负责甲基化修饰莱茵衣藻miRNA的功能，这对于揭示miRNA调控系统的起源和进化具有重要意义。