Active site mapping and substrate specificity of bacterial Hen1, a manganese-dependent 3' terminal RNA ribose 2'O-methyltransferase

Abstract

The RNA methyltransferase Hen1 and the RNA end-healing/sealing enzyme Pnkp comprise an RNA repair system encoded by an operon-like cassette present in bacteria from eight different phyla. Clostridium thermocellum Hen1 (CthHen1) is a manganese-dependent RNA ribose 2'O-methyltransferase that marks the 3' terminal nucleoside of broken RNAs and protects repair junctions from iterative damage by transesterifying endonucleases. Here we used the crystal structure of the homologous plant Hen1 to guide a mutational analysis of CthHen1, the results of which provide new insights to RNA end recognition and catalysis. We illuminated structure-activity relations at eight essential constituents of the active site implicated in binding the 3' dinucleotide of the RNA methyl acceptor (Arg273, Arg414), the manganese cofactor (Glu366, Glu369, His370, His418), and the AdoMet methyl donor (Asp291, Asp316). We investigated the effects of varying the terminal nucleobase, RNA size, RNA content, and RNA secondary structure on methyl acceptor activity. Key findings are as follows. CthHen1 displayed a fourfold preference for guanosine as the terminal nucleoside. RNA size had little impact in the range of 12-24 nucleotides, but activity declined sharply with a 9-mer. CthHen1 was adept at methylating a polynucleotide composed of 23 deoxyribonucleotides and one 3' terminal ribonucleotide, signifying that it has no strict RNA specificity beyond the 3' nucleoside. CthHen1 methylated RNA ends in the context of duplex secondary structures. These properties distinguish bacterial Hen1 from plant and metazoan homologs.

FIGURE 1.

Delineating the margins of the CthHen1 methyltransferase domain. (A) Aliquots (5 μg) of recombinant His₆-tagged WT CthHen1 and the truncated proteins spanning the amino acid residues as indicated were analyzed by SDS-PAGE. The Coomassie blue-stained gel is shown. The positions and sizes (kDa) of marker polypeptides are indicated on the left. (B) Methyltransferase reaction mixtures (10 μL) contained 20 μM [³H-CH₃]AdoMet, 10 μM (100 pmol) 24-mer RNA, and CthHen1 proteins as specified. The extents of ³H-methyl transfer to RNA are plotted as a function of input enzyme. (C) The tertiary structure of the C-terminal catalytic domain of CthHen1 is shown as a gray cartoon trace, starting from amino acid 268. The AdoHcy in the methyl donor site, depicted as a stick model, was imported from the structure of Anabaena variabilis Hen1 after superimposing it on the CthHen1 structure. The figure was prepared using coordinates from PDB entries 3JWG and 3JWH (Chan et al. 2009b).

FIGURE 2.

Conserved active site of plant and bacterial Hen1 methyltransferase domains. (A) Stereo view of the active site of Arabidopsis thaliana Hen1 (Huang et al. 2009) (from PDB 3HTX). AdoHcy (SAH) and the 3′ terminal dinucleotide of the RNA methyl acceptor are shown as stick models with gray carbons. The divalent cation is depicted as a magenta sphere; waters are red spheres. The amino acid residues contacting the methyl donor, the metal ion, and the RNA methyl acceptor are shown as stick models with beige carbons. The amino acids are labeled according to their CthHen1 equivalents. Atomic contacts are indicated by dashed lines. (B) The amino acid sequences of the Clostridium thermocellum (Cth) and Arabidopsis thaliana (Ath) Hen1 methyltransferase domains are aligned. Positions of side chain identity/similarity are indicated by a circle. The conserved AdoMet-binding motifs are shaded in yellow. Gaps in the alignment are indicated by a dashed line. A peptide segment of AthHen1 that is disordered in the crystal structure and missing from CthHen1 is highlighted in red font. The nine conserved amino acids that were subjected to mutational analyses in the present study are indicated by a vertical bar.

FIGURE 3.

Effects of alanine mutations on CthHen1 methyltransferase activity. (A) Aliquots (5 μg) of recombinant WT CthHen1-(259–465) and the indicated alanine mutants were analyzed by SDS-PAGE. The Coomassie blue-stained gel is shown. The positions and sizes (kDa) of marker polypeptides are indicated on the left. (B) Methyltransferase reaction mixtures contained 20 μM [³H-CH₃]AdoMet, 10 μM (100 pmol) 24-mer RNA, and 4 μM (40 pmol) CthHen1 proteins as specified. The extents of RNA methylation are plotted. Each datum is an average of three separate experiments ± SEM.

FIGURE 4.

Effects of conservative substitutions at essential residues. (A) Aliquots (5 μg) of recombinant WT CthHen1-(259–465) and the indicated mutants were analyzed by SDS-PAGE. The Coomassie blue-stained gel is shown. The positions and sizes (kDa) of marker polypeptides are indicated on the left. (B) Methyltransferase reaction mixtures contained 20 μM [³H-CH₃]AdoMet, 10 μM (100 pmol) 24-mer RNA, and 4 μM (40 pmol) CthHen1 proteins as specified. The extents of RNA methylation are plotted. Each datum is an average of three separate experiments ± SEM.

FIGURE 5.

CthHen1 prefers guanosine at the 3′-terminal position of the RNA methyl acceptor. Methyltransferase reaction mixtures contained 20 μM [³H-CH₃]AdoMet, 10 μM (100 pmol) 24-mer RNAs differing in their 3′-terminal nucleosides (G, U, A, or C), and CthHen1 as specified. The extents of RNA methylation of each substrate are plotted as a function of input CthHen1. Each datum is an average of three separate experiments ± SEM. The primary structures of the RNAs are shown below the graph. The 3′ terminal nucleobases are highlighted in gray.

FIGURE 6.

Effect of RNA length on methyl acceptor activity. Methyltransferase reaction mixtures contained 20 μM [³H-CH₃]AdoMet, 10 μM (100 pmol) 24-mer, 18-mer, 12-mer, or 9-mer RNAs, and CthHen1 as specified. The extents of RNA methylation are plotted as a function of input CthHen1. Each datum is an average of three separate experiments ± SEM. The primary structures of the RNAs are shown below the graph.

FIGURE 7.

A single ribonucleotide at 3′-terminus of a DNA strand suffices as a methyl acceptor. Methyltransferase reaction mixtures contained 20 μM [³H-CH₃]AdoMet, 10 μM (100 pmol) D23R1, D17R1, or D11R1 polynucleotides, and CthHen1 as specified. The extents of polynucleotide methylation are plotted as a function of input CthHen1. Each datum is an average of three separate experiments ± SEM. The primary structures of the substrates are shown below the graph.

FIGURE 8.

Effect of RNA secondary structure on methyl acceptor activity. Methyltransferase reaction mixtures contained 1 mM MnCl₂, 20 μM [³H-CH₃]AdoMet, 3 μM (30 pmol) CthHen1, and 10 μM (100 pmol) of 24-mer RNA strand (R24), either alone, hybridized to complementary DNA strands D20, D24, or D28, or mock-hybridized to a noncomplementary strand D24*. A control reaction mixture contained D24 in lieu of R24. The extents of polynucleotide methylation are plotted. Each datum is an average of two separate experiments; the bars denote the range. The structures of the R24 and DNA strands and the annealed duplexes are illustrated below the graph. Annealing was performed by mixing R24 with equimolar amounts of the DNA strand in 150 mM NaCl, then incubating the mixture for 10 min at 65°C, followed by 15 min at 37°C, and 15 min at 22°C. The annealed substrates were stored at −20°C and thawed immediately before use.