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Structural insights into mechanisms of the small RNA methyltransferase HEN1


RNA silencing is a conserved regulatory mechanism in fungi, plants and animals that regulates gene expression and defence against viruses and transgenes1. Small silencing RNAs of 20–30 nucleotides and their associated effector proteins, the Argonaute family proteins, are the central components in RNA silencing2. 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 nucleotide3,4,5,6. A conserved S-adenosyl-l-methionine-dependent RNA methyltransferase, HUA ENHANCER 1 (HEN1), and its homologues are responsible for this specific modification3,4,5,7,8. Here we report the 3.1 Å 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 Mg2+-dependent 2′-O-methylation mechanism.

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Figure 1: Structures of HEN1 in complex with a small RNA duplex and AdoHcy.
Figure 2: Small RNA substrate recognition by dsRBDs and LCD.
Figure 3: Small RNA substrate recognition by the MTase domain.
Figure 4: Proposed model for the specific recognition of small RNA substrates by HEN1 and the Mg 2+ -dependent 2′- O -methyltransferase mechanism.

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Protein Data Bank


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We thank K. Sergiy for assistance with the in-house X-ray generator operation, and the staff at Advanced Photon Source beamlines 19ID and 23ID, Argonne National Laboratory, for help with data collection. We thank T. Townes and H. Wang for critical reading of the manuscript. This work was supported by a start-up fund (to J.-B.M.) and partly by a grant from the V Foundation for Cancer Research (to J.-B.M.) and a grant from the National Science Foundation (MCB-0718029 to X.C.). D.G.V. is supported by grants from National Institutes of Health (R01 GM074252 and R01 GM074840).

Author Contributions Y.H. expressed and purified proteins, grew crystals, solved structure and wrote the manuscript. J.-B.M. collected data, solved structure, performed crosslinking assays and wrote the manuscript. L.J. performed small RNA methyltransferase assays and wrote the manuscript. Q.H. and D.G.V. were involved in data processing and refinement. X.C. analysed data and wrote the manuscript. The overall project management and manuscript preparation were by Y.H., X.C. and J.-B.M.

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Correspondence to Jin-Biao Ma.

Additional information

Atomic coordinates and structural factors for the reported crystal structure have been deposited in the Protein Data Bank under access code 3HTX.

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Supplementary Information

This file contains Supplementary Notes, Supplementary References, Supplementary Tables 1-3 and Supplementary Figures S1-S10 with Legends. (PDF 7558 kb)

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Huang, Y., Ji, L., Huang, Q. et al. Structural insights into mechanisms of the small RNA methyltransferase HEN1. Nature 461, 823–827 (2009).

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