Unpaired nucleotides on the stem of microRNA precursor are important for precise cleavage by Dicer‐like 1 in Arabidopsis
Unpaired nucleotides on the stem of microRNA precursor are important for precise cleavage by Dicer‐like 1 in Arabidopsis
复制标题
microRNA 前体茎上的不配对核苷酸对于拟南芥中 Dicer-like 1 的精确切割非常重要
DOI：
10.1111/gtc.12927
复制
发表时间：
2022
期刊：
影响因子：
2.1
通讯作者：
Yuji Iwata
中科院分区：
文献类型：
--
作者：
Rikako Hirata;Tomoya Makabe;Kei‐ichiro Mishiba;Nozomu Koizumi;Samir M. Hamdan;Yuji Iwata
DCL 1（Dicer-like 1）是植物microRNA（miRNA）生物发生机制的核心组成部分。miRNA被转录为前体RNA，称为初级miRNA（primarymiRNA），其被DCL 1分两步切割以产生miRNA/miRNA* 双链体。Pri‐miRNA是一种单链RNA，在其茎上形成具有许多未配对碱基（以下称为错配）的发夹结构。在本研究中，通过使用纯化的重组拟南芥DCL 1，我们提出的证据表明，错配的茎的pri-miRNA是重要的精确切割DCL 1。我们发现，在miRNA/miRNA* 双链体末端的环远端侧的错配对于体外有效切割pri-miRNA是重要的，正如之前在植物中所提出的那样。相反，远离miRNA/miRNA* 双链体区域的错配对于确定DCL 1的切割位置是重要的。纯化的DCL 1蛋白在野生型pri-miRNA变体通常不被切割的位置切割突变型pri-miRNA变体而没有这种错配，导致不同于miRNA的小RNA的积累增加。因此，我们的研究结果表明，除了与ssRNA-dsRNA连接的距离之外，pri-miRNA茎上的错配也是植物中DCL 1精确加工pri-miRNA的决定因素。
Dicer‐like 1 (DCL1) is a core component of the plant microRNA (miRNA) biogenesis machinery. MiRNA is transcribed as a precursor RNA, termed primary miRNA (pri‐miRNA), which is cleaved by DCL1 in two steps to generate miRNA/miRNA* duplex. Pri‐miRNA is a single‐stranded RNA that forms a hairpin structure with a number of unpaired bases, hereafter called mismatches, on its stem. In the present study, by using purified recombinant Arabidopsis DCL1, we presented evidence that mismatches on the stem of pri‐miRNA are important for precise DCL1 cleavage. We showed that a mismatch at the loop‐distal side of the end of miRNA/miRNA* duplex is important for efficient cleavage of pri‐miRNA in vitro, as previously suggested in planta. On the contrary, mismatches distant from the miRNA/miRNA* duplex region are important for determining the cleavage position by DCL1. The purified DCL1 proteins cleaved mutant pri‐miRNA variants without such mismatches at a position at which wild‐type pri‐miRNA variants are not usually cleaved, resulting in an increased accumulation of small RNA different from miRNA. Therefore, our results suggest that, in addition to the distance from the ssRNA‐dsRNA junction, mismatches on the stem of pri‐miRNA function as a determinant for precise processing of pri‐miRNA by DCL1 in plants.