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.

Figure 1.

Functional analysis of HEN1 domains. (A) Representation of domains on the structure of HEN1 in complex with the miR173/miR173* duplex (red) and AdoHcy (black) (23). (B) Schematic view of the HEN1 deletion variants. RNA binding was assessed in vitro by EMSA using the miR173/miR173* substrate; methyl transfer activity was studied by monitoring incorporation of [³H]-methyl groups from labeled AdoMet into miR173/miR173*. R¹ and R² (blue and purple, correspondingly): double-stranded RNA-binding domains; L (yellow): La-motif-containing domain; F (orange): FK506-binding protein-like domain; M (green): methyltransferase domain. (C) Positions of HEN1 mutations.

Figure 2.

Distinct contributions of the double-stranded RNA-binding domains R¹ and R² to miRNA/miRNA* binding. Left, the RNA-binding capacity of the truncated and mutation variants of HEN1 was assessed by EMSA using 0.25 μM protein and 0.05 μM miR173/miR173* duplex. Right, amino acid residues selected for mutagenesis in the dsRNA-binding domains R¹ (blue) and R² (purple).

Figure 3.

Interaction of the HEN1-RHK389,397,477AAA mutant with miR173/miR173* and siR173/siR173* substrates. (A) Native PAA gel EMSAs of 0.25 μM HEN1 or 7 μM HEN1-RHK389,397,477AAA binding to 0.05 μM unmethylated miR173/miR173*, fully methylated miR173^CH3/miR173*^CH3 and siR173/siR173* RNA duplexes in the absence (−) and presence (+) of 100 μM AdoHcy. wt: wild-type HEN1; RHK: mutant HEN1-RHK389,397,477AAA. (B), (C) Comparison of proteins’ affinity toward different RNA substrates. HEN1 (○) and mutant HEN1-RHK389,397,477AAA (•) mixed with 50 pM miR173/miR173* (B) or siR173/siR173* (C) RNA was incubated without (the left diagram) or with (the right diagram) 100 μM AdoHcy. Fits to a single-site saturation equation are shown as solid lines. (D), (E) Dissociation analysis of HEN1 (○) and its mutant HEN1-RHK389,397,477AAA (•) binary HEN1•RNA (the diagram at the left) and ternary HEN1•RNA•AdoHcy (the diagram at the right) complexes with miR173/miR173* (D) and siR173/siR173* (E) duplexes. Full-length HEN1 (0.25 μM) or mutant (7 μM) were incubated for 30 min with 0.15 nM ³²P-labeled RNA duplexes in the absence of the cofactor or in the presence of 100 μM AdoHcy. A 13 000-fold excess of competitor unlabeled RNA substrate was added and aliquots were withdrawn at specified time points for immediate analysis by EMSA. Decay time courses of the binary and ternary complexes along with single-exponential fits (dotted lines) or two-exponential fits (solid lines) are shown.

Figure 4.

HEN1 interacts with HYL1 in vitro and in vivo. (A) Titration of RNA•HYL1 with HEN1 reveals a high molecular weight complex. Varying amount of HEN1 (from 0.03 to 1 μM) was added to the RNA•HYL1 complex, formed after preincubation of 94 nM of protein with 45 nM of siR173/siR173*. (+) and (−) indicates presence and absence of particular proteins. (B) Validation of the interaction between HEN1 and HYL1 using a two-hybrid assay. Full-length cDNA of HEN1 and HYL1 were fused to the DNA encoding the LexA DNA-binding domain (BD) and B42 transcriptional activation domain (AD), respectively. Four independent co-transformants in yeast strain EGY48 were screened for the lacZ and Leu reporter genes on plates containing selective X-Gal medium in the absence (Gal/–Leu) or with leucine (Gal/+Leu). (C) Analysis of protein–protein interaction among HYL1 and HEN1 domains by GST pull-down assay. The experiments were performed using 68 pmol of GST or GST-fused HYL1 (GST-HYL1) and 136 pmol of His-tagged HEN1 proteins. The pull-down fractions were analyzed by protein blotting with anti-His antibodies. Presence of GST-HYL1 and GST proteins in pull-down samples was confirmed by western blot with anti-GST antibodies. The input fractions represent 20% of the total amount of His-tagged proteins used in pull-down assays. Schemes of truncated HEN1 proteins are depicted in Figure 1. HEN1-mut and FM-mut: the full-length HEN1 and truncated protein composed of HEN1 F (FK506-binding protein-like) and M (methyltransferase) domains, respectively, which have three hydrophobic residues V543, L544 and V550 changed to alanine.

Figure 5.

HYL1 interacts with HEN1 via its second dsRNA-binding domain R². (A) GST pull-down assays indicating HEN1 binds to HYL1-R¹R² and R² but not to HYL1-R¹. On the right schematic representation of full-length HYL1 and shorter proteins of separate domains is given. R¹ and R²: double-stranded RNA-binding domains, NLS: nuclear localization signal. (B) Two-hybrid interaction between FK506-binding protein-like domain HEN1-F and the second dsRNA-binding domain HYL1-R². All experiments were performed as described in Figure 4.

Figure 6.

Lack of detectable interactions between SERRATE and HEN1 methyltransferase. (A) Yeast two-hybrid analysis revealed no in-cell interactions between HEN1 and SERRATE (SE). HYL1, a known SE-core-interacting partner (6), was used as a positive control for yeast assay. (B) GST pull-down experiment shows no interaction between central part of SERRATE (SE-core) and full-length HEN1 proteins in vitro. Experiments were carried out as depicted in Figure 4. Schematic representation of the domains in SE is made according to Machida et al. (6). SE-core construct is comprised of N-terminal (N), middle (Mid) and Zinc finger domains.

Figure 7.

Interaction of HEN1 with individual domains of DICER-LIKE 1 (DCL1). (A) Yeast two-hybrid analysis of the interaction between HEN1 and DCL1 domains. Abbreviations of domains are as follows: NLS: nuclear localization signal, Helicase: DExD/H-box RNA helicase, DUF283: domain of unknown function 283, PAZ: Piwi/Argonaute/Zwille domain, RNase III: ribonuclease III, R¹, R²: dsRNA-binding domains. Interactions between SE-core with Helicase and HYL1 with DUF283 or R¹R² domains of DCL1 served as positive controls (5,6,31). (B) Detection of the interaction between DCL1 domains and HEN1 using GST pull-down. GST-Helicase, GST-DUF283, GST-PAZ and GST-R¹R² denote GST-DCL1-Helicase, GST-DCL1-DUF283, GST-DCL1-PAZ and GST-DCL1-R¹R², respectively. Experiments were carried out as described in Figure 4.

Figure 8.

HEN1 interaction network and the proposed model of late stages of miRNA biogenesis. (A) Protein–protein interaction network involving SE, HYL1, DCL1 and HEN1. Cyan lines show interactions experimentally determined in this work, purple and black lines depict those reported previously (6,31,32). (B) Proposed model of miRNA biogenesis envisions that after the miRNA/miRNA* duplex is cut out of its precursor, SE is expelled and HEN1 methyltransferase is bound in the microprocessor complex to form a HYL1•HEN1•DCL1 complex, which might represent the still unidentified plant RISC-loading complex (RLC) anticipated by Eamens et al. (20). We hypothesize that this complex could direct HEN1 methylation (red circle) to the target miRNA strand (black) thus labeling it for incorporation into AGO1 complex.