Functional mapping of the plant small RNA methyltransferase: HEN1 physically interacts with HYL1 and DICER-LIKE 1 proteins

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Arabidopsis Proteins / chemistry
  • Arabidopsis Proteins / genetics
  • Arabidopsis Proteins / metabolism*
  • Cell Cycle Proteins / chemistry
  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism*
  • Electrophoretic Mobility Shift Assay
  • Methylation
  • Methyltransferases / chemistry
  • Methyltransferases / genetics
  • Methyltransferases / metabolism*
  • MicroRNAs / chemistry
  • MicroRNAs / genetics
  • MicroRNAs / metabolism
  • Models, Molecular
  • Molecular Sequence Data
  • Mutation
  • Nucleic Acid Conformation
  • Protein Binding
  • Protein Interaction Mapping
  • Protein Structure, Tertiary
  • RNA, Plant / chemistry
  • RNA, Plant / genetics
  • RNA, Plant / metabolism
  • RNA-Binding Proteins / chemistry
  • RNA-Binding Proteins / genetics
  • RNA-Binding Proteins / metabolism*
  • Ribonuclease III / chemistry
  • Ribonuclease III / genetics
  • Ribonuclease III / metabolism*
  • Sequence Homology, Amino Acid
  • Two-Hybrid System Techniques


  • Arabidopsis Proteins
  • Cell Cycle Proteins
  • HEN1 protein, Arabidopsis
  • HYL1 protein, Arabidopsis
  • MicroRNAs
  • RNA, Plant
  • RNA-Binding Proteins
  • Methyltransferases
  • RNA 2'-O-methyltransferase
  • DCL1 protein, Arabidopsis
  • Ribonuclease III