title: Functional mapping of the plant small RNA methyltransferase: HEN1 physically interacts with HYL1 and DICER-LIKE 1 proteins.
abstract: 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.
authors: Department of Biological DNA Modification, Institute of Biotechnology, Vilnius University, Vilnius LT-02241, Lithuania.
authors: Simona
authors: S
authors: Baranauskė
authors: Department of Biological DNA Modification, Institute of Biotechnology, Vilnius University, Vilnius LT-02241, Lithuania.
authors: Milda
authors: M
authors: Mickutė
authors: Department of Biological DNA Modification, Institute of Biotechnology, Vilnius University, Vilnius LT-02241, Lithuania.
authors: Alexandra
authors: A
authors: Plotnikova
authors: Department of Plant Breeding and Genetics, Max Planck Institute for Plant Breeding Research, 50829 Cologne, Germany.
authors: Andreas
authors: A
authors: Finke
authors: Department of Bioinformatics, Institute of Biotechnology, Vilnius University, Vilnius LT-02241, Lithuania.
authors: Česlovas
authors: Č
authors: Venclovas
authors: Department of Biological DNA Modification, Institute of Biotechnology, Vilnius University, Vilnius LT-02241, Lithuania.
authors: Saulius
authors: S
authors: Klimašauskas
authors: Department of Biological DNA Modification, Institute of Biotechnology, Vilnius University, Vilnius LT-02241, Lithuania giedrius@ibt.lt.
authors: Giedrius
authors: G
authors: Vilkaitis
mesh_terms: D000595:Amino Acid Sequence; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D008745:Methylation; D008780:Methyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D035683:MicroRNAs / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D018749:RNA, Plant / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D043244:Ribonuclease III / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D020798:Two-Hybrid System Techniques
chemical_list: D029681:Arabidopsis Proteins; D018797:Cell Cycle Proteins; C450825:HEN1 protein, Arabidopsis; C420966:HYL1 protein, Arabidopsis; D035683:MicroRNAs; D018749:RNA, Plant; D016601:RNA-Binding Proteins; D008780:Methyltransferases; C052744:RNA 2'-O-methyltransferase; C402383:DCL1 protein, Arabidopsis; D043244:Ribonuclease III