Department of Cell and Developmental Biology, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK.
Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, DK-2200, Copenhagen N, Denmark.
Nucleic Acids Res. 2020 Sep 4;48(15):8767-8781. doi: 10.1093/nar/gkaa577.
MicroRNA (miRNA)-mediated cleavage is involved in numerous essential cellular pathways. miRNAs recognize target RNAs via sequence complementarity. In addition to complementarity, in vitro and in silico studies have suggested that RNA structure may influence the accessibility of mRNAs to miRNA-induced silencing complexes (miRISCs), thereby affecting RNA silencing. However, the regulatory mechanism of mRNA structure in miRNA cleavage remains elusive. We investigated the role of in vivo RNA secondary structure in miRNA cleavage by developing the new CAP-STRUCTURE-seq method to capture the intact mRNA structurome in Arabidopsis thaliana. This approach revealed that miRNA target sites were not structurally accessible for miRISC binding prior to cleavage in vivo. Instead, we found that the unfolding of the target site structure plays a key role in miRISC activity in vivo. We found that the single-strandedness of the two nucleotides immediately downstream of the target site, named Target Adjacent nucleotide Motif, can promote miRNA cleavage but not miRNA binding, thus decoupling target site binding from cleavage. Our findings demonstrate that mRNA structure in vivo can modulate miRNA cleavage, providing evidence of mRNA structure-dependent regulation of biological processes.
微小 RNA (miRNA)-介导的切割参与了许多重要的细胞途径。miRNA 通过序列互补来识别靶 RNA。除了互补性，体外和计算机模拟研究表明，RNA 结构可能会影响 mRNA 对 miRNA 诱导的沉默复合物 (miRISCs) 的可及性，从而影响 RNA 沉默。然而，mRNA 结构在 miRNA 切割中的调节机制仍不清楚。我们通过开发新的 CAP-STRUCTURE-seq 方法来研究体内 RNA 二级结构在 miRNA 切割中的作用，该方法可捕获拟南芥中完整的 mRNA 结构组。该方法表明，miRNA 靶位点在体内切割之前并没有结构上可用于 miRISC 结合。相反，我们发现靶位点结构的展开在 miRISC 体内活性中起着关键作用。我们发现靶位点下游的两个核苷酸的单链状态，称为靶相邻核苷酸基序，可促进 miRNA 切割，但不促进 miRNA 结合，从而将靶位点结合与切割分离。我们的发现表明，体内 mRNA 结构可以调节 miRNA 切割，为依赖于 mRNA 结构的生物过程调节提供了证据。
