Baranauskė Simona, Mickutė Milda, Plotnikova Alexandra, Finke Andreas, Venclovas Česlovas, Klimašauskas Saulius, Vilkaitis Giedrius
Department of Biological DNA Modification, Institute of Biotechnology, Vilnius University, Vilnius LT-02241, Lithuania.
Department of Plant Breeding and Genetics, Max Planck Institute for Plant Breeding Research, 50829 Cologne, Germany.
Nucleic Acids Res. 2015 Mar 11;43(5):2802-12. doi: 10.1093/nar/gkv102. Epub 2015 Feb 12.
Methylation of 3'-terminal nucleotides of miRNA/miRNA* is part of miRNAs biogenesis in plants but is not found in animals. In Arabidopsis thaliana this reaction is carried out by a multidomain AdoMet-dependent 2'-O-methyltransferase HEN1. Using deletion and structure-guided mutational analysis, we show that the double-stranded RNA-binding domains R(1) and R(2) of HEN1 make significant but uneven contributions to substrate RNA binding, and map residues in each domain responsible for this function. Using GST pull-down assays and yeast two-hybrid analysis we demonstrate direct HEN1 interactions, mediated by its FK506-binding protein-like domain and R(2) domain, with the microRNA biogenesis protein HYL1. Furthermore, we find that HEN1 forms a complex with DICER-LIKE 1 (DCL1) ribonuclease, another key protein involved in miRNA biogenesis machinery. In contrast, no direct interaction is detectable between HEN1 and SERRATE. On the basis of these findings, we propose a mechanism of plant miRNA maturation which involves binding of the HEN1 methyltransferase to the DCL1•HYL1•miRNA complex excluding the SERRATE protein.
微小RNA（miRNA）/微小RNA* 3'末端核苷酸的甲基化是植物中miRNA生物合成的一部分，但在动物中未发现。在拟南芥中，该反应由多结构域依赖腺苷甲硫氨酸的2'-O-甲基转移酶HEN1进行。通过缺失和结构引导的突变分析，我们发现HEN1的双链RNA结合结构域R(1)和R(2)对底物RNA结合有显著但不均衡的贡献，并确定了每个结构域中负责此功能的残基。使用谷胱甘肽S-转移酶（GST）下拉实验和酵母双杂交分析，我们证明了HEN1通过其FK506结合蛋白样结构域和R(2)结构域与微小RNA生物合成蛋白HYL1直接相互作用。此外，我们发现HEN1与Dicer样1（DCL1）核糖核酸酶形成复合物，DCL1是参与miRNA生物合成机制的另一个关键蛋白。相比之下，未检测到HEN1与锯齿蛋白（SERRATE）之间有直接相互作用。基于这些发现，我们提出了一种植物miRNA成熟机制，该机制涉及HEN1甲基转移酶与DCL1•HYL1•miRNA复合物结合，排除锯齿蛋白。
