SMA1调控拟南芥miRNA合成的分子机制研究

miRNAs are a class of universal non-coding RNA in eukaryotes with the size of 21-24 nt. Plant miRNAs play essential regulatory roles in plant developmental, biotic and abiotic stress responses by repressing the expression of their target mRNAs. H​owever, the detailed molecular mechanisms on how miRNAs generate are still largely unknown in plants. Our preliminary study isolated a mutant with deduced miRNA level, named sma1. By map-based cloning, we identified SMA1 gene, that encodes an unknown protein with a DEAD domain. The ChIP results indicate that SMA1 protein associates with MIR gene promoter regions and promotes RNA polymerase II-dependent MIR transcription. Moreover, the results of BiFC and CoIP indicate that SMA1 interacts with DCL1 complex. I​n addition, we found that SMA1 regulates DCL1 pre-mRNA splicing and controls DCL1 protein level. Our findings suggest SMA1 should be involved in miRNA biogenesis. The current project is aimed to illuminate the molecular mechanisms on SMA1 regulating miRNA biogenesis using the techniques of Northern blot, small-RNA sequencing, protein interaction, protein activity detection, and genetic network analysis. The results obtained will provide novel insights into the miRNA biogenesis, thus lay the theoretical basis for the fine-tune of plant development and the improvement of agronomy.

miRNAs是真核生物中普遍存在的21-24nt的非编码RNA。植物miRNA通过抑制靶基因表达水平调控生长发育、生物和非生物胁迫等生物学过程。然而调控植物miRNA合成的分子机制并不十分清楚。申请人前期的研究分离了一个miRNA水平显著降低的突变体sma1，并​通过图位克隆技术鉴定了SMA1基因，其编码一个含有DEAD结构域的未知功能蛋白。ChIP实验结果表明，SMA1蛋白结合MIR基因的启动子区，并促进MIR基因的转录激活。BiFC和CoIP结果表明，SMA1蛋白直接与DCL1复合体互作。此外，SMA1调控DCL1 内含子剪切并影响蛋白表达水平。这些结果初步表明，SMA1参与miRNA的合成。 本项目拟通过Northern 杂交、小RNA 测序、蛋白互作分析、蛋白活性检测、 遗传网络分析等技术，阐明SMA1调控miRNA合成的分子机制，为精细调节植物生长发育和改善农业性状提供理论基础。

miRNAs调控植物生长发育、生物和非生物胁迫等重要生物学过程。然而调控植物miRNA合成的分子机制并不十分清楚。本项目借助于miRNA水平显著降低的突变体sma1-1，揭示了SMA1基因在调控植物miRNA合成过程中的重要功能。通过项目实施，明确了SMA1通过影响MIR启动子的转录活性调控MIR基因的转录水平；发现SMA1与miRNA合成的关键组分DCL1复合体直接互作，提供了SMA1参与miRNA加工的遗传和分子生物学证据；揭示SMA1调控pri-miRNA和pre-mRNA内含子可变剪切的重要作用；通过正向和反向遗传学获得了一系列SMA1相关候选蛋白因子和遗传材料。本项目的实施为理解植物SMA1的重要生物学功能奠定了坚实的基础。