Structural and biochemical insights into 2′-O-methylation at the 3′-terminal nucleotide of RNA by Hen1

doi:10.1073/pnas.0907540106

Structural and biochemical insights into 2′-O-methylation at the 3′-terminal nucleotide of RNA by Hen1

^aDepartment of Biochemistry and
^cCenter for Biophysics and Computational Biology, University of Illinois at Urbana–Champaign, Urbana, IL 61801; and
^bLife Science Collaborative Access Team, Argonne National Laboratory, Argonne, IL 60439

Edited by Jennifer A. Doudna, University of California, Berkeley, CA, and approved August 27, 2009
↵¹C.M.C. and C.Z. contributed equally to this work. (received for review July 7, 2009)

Abstract

Small RNAs of ≈20–30 nt have diverse and important biological roles in eukaryotic organisms. After being generated by Dicer or Piwi proteins, all small RNAs in plants and a subset of small RNAs in animals are further modified at their 3′-terminal nucleotides via 2′-O-methylation, carried out by the S-adenosylmethionine-dependent methyltransferase (MTase) Hen1. Methylation at the 3′ terminus is vital for biological functions of these small RNAs. Here, we report four crystal structures of the MTase domain of a bacterial homolog of Hen1 from Clostridium thermocellum and Anabaena variabilis, which are enzymatically indistinguishable from the eukaryotic Hen1 in their ability to methylate small single-stranded RNAs. The structures reveal that, in addition to the core fold of the MTase domain shared by other RNA and DNA MTases, the MTase domain of Hen1 possesses a motif and a domain that are highly conserved and are unique to Hen1. The unique motif and domain are likely to be involved in RNA substrate recognition and catalysis. The structures allowed us to construct a docking model of an RNA substrate bound to the MTase domain of bacterial Hen1, which is likely similar to that of the eukaryotic counterpart. The model, supported by mutational studies, provides insight into RNA substrate specificity and catalytic mechanism of Hen1.

Footnotes

²To whom correspondence should be addressed. E-mail: huang{at}uiuc.edu
Author contributions: C.M.C., C.Z., and R.H.H. designed research; C.M.C., C.Z., and J.S.B. performed research; C.M.C., C.Z., J.S.B., and R.H.H. analyzed data; and R.H.H. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.rcsb.org (PDB ID codes 3JWG, 3JWH, 3JWI, and 3JWJ).
This article contains supporting information online at www.pnas.org/cgi/content/full/0907540106/DCSupplemental.