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Nucleic Acids Res. 2015 Mar 11;43(5):2802-12. doi: 10.1093/nar/gkv102. Epub 2015 Feb 12.

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

Author information

1
Department of Biological DNA Modification, Institute of Biotechnology, Vilnius University, Vilnius LT-02241, Lithuania.
2
Department of Plant Breeding and Genetics, Max Planck Institute for Plant Breeding Research, 50829 Cologne, Germany.
3
Department of Bioinformatics, Institute of Biotechnology, Vilnius University, Vilnius LT-02241, Lithuania.
4
Department of Biological DNA Modification, Institute of Biotechnology, Vilnius University, Vilnius LT-02241, Lithuania giedrius@ibt.lt.

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.

PMID:
25680966
PMCID:
PMC4357707
DOI:
10.1093/nar/gkv102
[Indexed for MEDLINE]
Free PMC Article

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