Structural insights into mechanisms of the small RNA methyltransferase HEN1

Abstract

RNA silencing is a conserved regulatory mechanism in fungi, plants and animals that regulates gene expression and defence against viruses and transgenes. Small silencing RNAs of approximately 20-30 nucleotides and their associated effector proteins, the Argonaute family proteins, are the central components in RNA silencing. A subset of small RNAs, such as microRNAs and small interfering RNAs (siRNAs) in plants, Piwi-interacting RNAs in animals and siRNAs in Drosophila, requires an additional crucial step for their maturation; that is, 2'-O-methylation on the 3' terminal nucleotide. A conserved S-adenosyl-l-methionine-dependent RNA methyltransferase, HUA ENHANCER 1 (HEN1), and its homologues are responsible for this specific modification. Here we report the 3.1 A crystal structure of full-length HEN1 from Arabidopsis in complex with a 22-nucleotide small RNA duplex and cofactor product S-adenosyl-l-homocysteine. Highly cooperative recognition of the small RNA substrate by multiple RNA binding domains and the methyltransferase domain in HEN1 measures the length of the RNA duplex and determines the substrate specificity. Metal ion coordination by both 2' and 3' hydroxyls on the 3'-terminal nucleotide and four invariant residues in the active site of the methyltransferase domain suggests a novel Mg(2+)-dependent 2'-O-methylation mechanism.

Figure 1. Structures of HEN1 in complex with a small RNA duplex and AdoHcy

a, Ribbon diagram of the complex. dsRBD, violet; La motif, chocolate; LCD, wheat; dsRDB2, cyan; PLD, purple; MTase, green; linkers including L1, L2 and L4, grey; RNA strand to be methylated (m strand), red; RNA strand not to be methylated (u strand), blue; AdoHcy, yellow; Mg²⁺, brown. b, Ribbon diagram of the complex rotated by 90° about the horizontal axis relative to a. c, d, Surface and surface charge views of HEN1 in the complex in the same orientation as a. e, Schematic representation of the domains in HEN1 with the same colour codes as in a. The disordered L3 is represented by the dashed line. f, Sequences of the small RNA duplex used in the co-crystallization. The m strand and u strand are coloured red and blue, respectively, and the two termini, [5′-m:3′-u] and [5′-u:3′-m], are indicated.

Figure 2. Small RNA substrate recognition by dsRBDs and LCD

a, The duplex region of the small RNA substrate is bound by three RNA binding motifs in dsRBD1. b, The duplex region of the small RNA substrate is bound by two RNA binding motifs (RBM1 and RBM3) in dsRBD2. c, The LCD binds to the [5′-m:3′-u] terminus of the small RNA substrate. The 2-nucleotide 3′ overhang of the u strand is recognized by the La motif. The base of the 5′-terminal nucleotide G1_m is end-capped by W333 in the C-terminal LCD.

Figure 3. Small RNA substrate recognition by the MTase domain

a, The phosphate connecting the 2-nucleotide 3′-overhang of the m strand with the duplex region is specifically recognized by a conserved loop (F692–L697). The penultimate nucleotide A21_m is flipped out and its base is stacked on the side chains of L835 and R856. The phosphate of the 2-nucleotide overhang is hydrogen bonded by R701 and R856. b, The base of the 3′-terminal nucleotide of the m strand G22_m is stacked on the terminal base pair formed by A20_m and U1_u and the 5′-phosphate of the u strand is recognized by S747. c, Both 2′ and 3′ hydroxyls of the 3′-terminal nucleotide G22_m are coordinated to Mg²⁺ along with four invariant residues, E796, E799, H800 and H860. d, A stereo view of the Mg²⁺ coordination covered with F_o−F_c electron density omit map contoured at 3.0σ.

Figure 4. Proposed model for the specific recognition of small RNA substrates by HEN1 and the Mg²⁺-dependent 2′-O-methyltransferase mechanism

A small RNA substrate is targeted by multiple RNA binding domains in HEN1. The duplex region is gripped by dsRBD1 and dsRBD2, and one terminus is projected towards the MTase domain that is located within a range of 18–22 bp from another terminus end-capped by a tryptophan residue in LCD. Consequently, the MTase domain preferably recognizes the 2-nucleotide 3′ overhang on the small RNA substrate of 20–24 nucleotides in length and methylates the 2′-hydroxyl of the 3′-terminal nucleotide in a Mg²⁺-dependent manner.