The structure of the endoribonuclease XendoU: From small nucleolar RNA processing to severe acute respiratory syndrome coronavirus replication

Abstract

Small nucleolar RNAs (snoRNAs) play a key role in eukaryotic ribosome biogenesis. In most cases, snoRNAs are encoded in introns and are released through the splicing reaction. Some snoRNAs are, instead, produced by an alternative pathway consisting of endonucleolytic processing of pre-mRNA. XendoU, the endoribonuclease responsible for this activity, is a U-specific, metal-dependent enzyme that releases products with 2'-3' cyclic phosphate termini. XendoU is broadly conserved among eukaryotes, and it is a genetic marker of nidoviruses, including the severe acute respiratory syndrome coronavirus, where it is essential for replication and transcription. We have determined by crystallography the structure of XendoU that, by refined search methodologies, appears to display a unique fold. Based on sequence conservation, mutagenesis, and docking simulations, we have identified the active site. The conserved structural determinants of this site may provide a framework for attempting to design antiviral drugs to interfere with the infectious nidovirus life cycle.

Fig. 1.

The structure of XendoU. (Upper) Secondary structure of XendoU along the amino acid sequence. Secondary structure assignment is according to an algorithm implemented in PyMol. Colors change from blue to red going from the N to the C terminus. Residues not identified in the map are in lower case. Residues in italics have been mutated (12) as follows: dark yellow, nearly silent mutation; red, cleavage inactivation in single and double (E161Q+E167Q) mutants. T278 was mutated as described in Fig. 3. (Lower) (a) Cartoon representation of the overall fold of XendoU. The color code is as specified above. Helices are numbered from 1 to 9 and β-strands from 1 to 8. β-sheet I is formed by β1 and β2; β-sheet II by β3, β4, and β5; β-sheet III by β6, β7, and β8. (b) The α7 helix position is restrained. Conserved hydrophobic residues in the α7 helix form a hydrophobic cluster with residues belonging to α3, α4, α6, and α9 helices and β-sheet III. A H-bond between G166 and G176 (at the top) seals the ends of the loop 166–179. (c) Docking of the 3′ UMP anchored onto the phosphate-binding site. The 3′ UMP and the interacting residues are represented. In addition to the residues binding the phosphate, the conserved H178, Y280, and G176 (which appear to be in contact with the base) and S157 and Y147 (which may stabilize a proton on the Nε of H162) are shown. K224, E161, and E167 are also shown. (d) The 2Fo − Fc map contoured at 1.2σ on the phosphate-binding site. The phosphate is bound to residues H162, H272, N270, and T278 and to the positively charged R149 in one of its conformations.

Fig. 2.

Partial alignment of XendoU sequence (residues 131–285) with homologs from eukaryotes, nidoviruses, and a cyanobacterium. Alignment was performed by ClustalW (www.ebi.ac.uk/clustalw) and manually modified for consistency with the secondary structure prediction (29). Gaps were allowed in loops, and insertions are indicated by the total number of residues (to improve readability). Amino acid color codes are as follows: red, H; yellow, P/G; brown, hydrophobic; blue, R/K; green, D/E/N/Q; magenta, S/T/C. The top line indicates the secondary structure of XendoU. Residues forming the 3′ UMP-binding site (described in Fig. 1d) are indicated by arrowheads. An abbreviated name of the organism and the number of the first amino acid of the aligned region are indicated. The corresponding data bank codes are as follows: Vertebrata: (1) Homo sapiens M32402, (2) Pan troglodytes XP_522366, (3) Mus musculus NP_032928, (4) Gallus gallus XM428848, (5) Xenopus laevis AJ507315, (6) Paralichtys olivaceus BAA88246; Insecta: (7) Drosophila melanogaster AAF47979, (8) Anopheles gambiae XP311978; Nematoda: (9) Caenorhabditis elegans NP_492590; Planta: (10) Arabidopsis thaliana gi 25407557; Cyanobacteria: (11) Nostoc punctiforme NZ_AAAY02000132; Nidovirales-Coronaviridae: (12) SARS CoV NC_004718, (13) human CoV group 1 strain 229E NC_002645, (14) bovine CoV group2 NC003045, (15) murine hepatitis CoV strain A59 NC_001846, (16) avian infectious bronchitis CoV NC_001451, (17) equine Berne torovirus X52374; Nidovirales-Arteriviridae: (18) Lelystad arterivirus or porcine reproductive and respiratory syndrome virus M96262, (19) equine arteritis arterivirus X53459; Nidovirales-Roniviridae: (20) gill-associated factor okavirus AF227196.

Fig. 3.

Binding and processing activity of T278A mutant. (a) XendoU mutant can bind the RNA in a gel mobility-shift assay. Two femtomoles of ³²P-labeled U16 snoRNA precursor were incubated with His-XendoU or its mutant T278A at a concentration of 0.05 μM (lanes 2) or 0.1 μM (lanes 3); nonincubated RNA was loaded in lane 1 as control. C points to the RNA/protein complex. (b) In vitro processing assay. The ³²P-labeled U16 snoRNA precursor was incubated with 50 ng of His-XendoU (lanes WT) or T278A mutant (lanes T278A) for 10 min (lanes 2) or 20 min (lanes 3). As a control, nonincubated RNA was loaded in lane 1. The processing products are schematized at the left: The U16-containing precursor is indicated by P. Cleavage upstream to U16 produces the I-2 and the complementary cut-off molecule I-2′, whereas cleavage downstream generates the I-3 and I-3′ products. Double cleavage produces pre-U16 molecules. The intron is shown as a continuous line and the U16 snoRNA coding region as a black box.