植物miRNA的产生主要通过以DCL1为核心的复合物介导。已知miRNA的产生受到了前体mRNA拼接过程的调控，但其分子机制并不清楚。申请人前期的工作表明负责降解拼接副产物-内含子形成的套索RNA的去分支酶（DBR1）全局性的调节了植物miRNA的产生。申请人发现dbr1突变体中全基因组范围内的miRNA水平降低，但初级miRNA (pri-miRNA) 却积累，而且pri-miRNA与DCL1的结合效率显著下降。因此我们推测DBR1介导的套索RNA的降解对miRNA产生至关重要。本课题将深入研究DBR1促进DCL1结合pri-miRNA的分子机理；解析DBR1和套索RNA的表达调控机制；鉴定DBR1和套索RNA的结合蛋白；阐明套索RNA代谢与miRNA产生途径互作的分子基础。该项目的完成将拓展人们对套索RNA功能的崭新认识，为诠释miRNA产生与mRNA拼接的互作机制开辟新视野。

MicroRNAs (miRNAs) play an important role in multiple biological processes, miRNA biogenesis begins with the processing of primary miRNAs (pri-miRNAs), which contain a hairpin structure that is cleaved twice by the DCL1 complex. Splicing of mRNA precursors (pre-mRNA) is a critical step in gene expression in eukaryotes. Studies in the past decade years have established active crosstalk between miRNA biogenesis and pre-mRNA splicing. However, the mechanisms underlying the interplay between these two processes are still unclear. As you know, lariat RNAs are formed from cleaved intron with a circular loop and a linear tail during pre-mRNA splicing, which are usually thought as by-products of splicing and should be quickly linearized by the debranching enzyme (DBR1) in eukaryotes. Null mutants of DBR1 are embryo lethal in both plants and animals, but why the accumulation of lariat RNAs is intolerant remains unexplored. In a genetic screening aiming to identify new players in miRNA biogenesis, we isolated a weak and viable mutant allele of DBR1 in Arabidopsis. We found that lariat RNAs over-accumulate concomitantly with global reduction of miRNAs in the dbr1 mutant. Pri-miRNA is accumulated in the dbr1 mutant, and the binding of the DCL1 complex with pri-miRNAs was greatly reduced. Circular RNA seq analysis identifies that hundreds of lariat RNAs exist in wild type plants, and thousands of lariat RNAs accumulate in the dbr1 mutant. RIP assay shows that the DCL1 complex can bind lariat RNAs, and R-EMSA assay further shows that circular RNAs competitively inhibit the binding of the DCL1 complex with pri-miRNAs. We thus propose that lariat RNAs act as decoys to inhibit miRNA biogenesis in plants, and this function could contribute to the embryo lethality in dbr1 mutants. Thus, in this proposed study, we will focus on how DBR1 promotes DCL1 binding to pri-miRNA; how DBR1 and lariat RNA are regulated; how lariat RNA itself is regulated, and what components of DBR1 and lariat RNAs bind. The accomplishment of this study will not only broaden our knowledge on the function of lariat RNAs and also open a new window for us to understand how miRNA biogenesis interplays with pre-mRNA splicing.