The yeast ribosome synthesis factor Emg1 is a novel member of the superfamily of alpha/beta knot fold methyltransferases

Abstract

Emg1 was previously shown to be required for maturation of the 18S rRNA and biogenesis of the 40S ribosomal subunit. Here we report the determination of the crystal structure of Emg1 at 2 A resolution in complex with the methyl donor, S-adenosyl-methionine (SAM). This structure identifies Emg1 as a novel member of the alpha/beta knot fold methyltransferase (SPOUT) superfamily. In addition to the conserved SPOUT core, Emg1 has two unique domains that form an extended surface, which we predict to be involved in binding of RNA substrates. A point mutation within a basic patch on this surface almost completely abolished RNA binding in vitro. Three point mutations designed to disrupt the interaction of Emg1 with SAM each caused>100-fold reduction in SAM binding in vitro. Expression of only Emg1 with these mutations could support growth and apparently normal ribosome biogenesis in strains genetically depleted of Emg1. We conclude that the catalytic activity of Emg1 is not essential and that the presence of the protein is both necessary and sufficient for ribosome biogenesis.

Figure 1.

The structure of Emg1. (A) Topology diagram of the Emg1 protein. The secondary structure of the core alpha/beta knot fold of the methyltransferase is colored in red and the Emg1-specific insertions are in blue. The 3–10 helices are indicated by the capital letter H, while canonical helices and strands are indicated by Greek letters. (B) Ribbon representation of the Emg1 protein with the same color code as panel A. The SAM is shown in stick representation. The right view is turned by 180° in respect to the left view. (C) Surface representation of the Emg1 protein. The surface is colored in shades of red corresponding to sequence conservation, from white (less conserved) to dark red (most conserved). Only eukaryote sequences were included in the determination of the degree of conservation [calculated with rate4site (31)]. The two orientations are identical to those shown in panel B and the SAM is represented as sticks. (D) Electrostatic potential surface representation of the Emg1 protein. The two orientations are identical to panel B and C and the SAM is represented as sticks. The Emg1-specific insertion (blue in panel B) is highly conserved (panel C) and displays a strong positive surface, ideal for RNA binding. Two sulfates bound to this surface are shown in stick representation. The potential was calculated using MEAD (32).

Figure 2.

Emg1 SAM-binding site. Stereo representation of the SAM-binding site. SAM is shown in yellow. The most relevant residues of Emg1 are shown in stick representation. Hydrogen bonds are shown in dashed lines. The numbering of residues mutated below is colored in red. The conformation of Asp214 in the apo protein is shown in blue sticks.

Figure 3.

Sequence alignment of Emg1 proteins of two different phylogenetic groups. (1) Eukaryotes and (2) Archaea. The secondary structure of Emg1 is shown above the alignment. Residues mutated in the SAM-binding site are indicated with a triangle. The figure was generated with a special version of ESPript (33).

Figure 4.

Emg1 dimerizes and directly binds RNA. (A) Emg1 binds RNA directly. In vitro transcribed, [³²P]-labeled RNA was incubated with HisZZ-tagged wild-type (WT) or R88D Emg1 and retrieved on IgG Sepharose. RNA was extracted from eluates, separated on a 6% polyacrylamide/urea gel and analyzed by autoradiography and phosphoimaging. An autoradiograph is shown with relative intensities measured by phosphoimager indicated below. While the WT protein binds RNA efficiently, binding is strongly compromised for the R88D mutant. (B) Dimerization of Emg1 analyzed by binding experiment. Recombinant HisZZ-tagged WT (lanes 1–3) or R88D Emg1 (lanes 4–6) was incubated with His-tagged Emg1 and retrieved on IgG Sepharose. Eluted samples were analyzed after SDS–PAGE and Coomassie staining, with inputs shown on the right. The HisZZ-tagged WT Emg1 pulls down the His-tagged fusion, showing dimerization of Emg1. The R88D mutation reduces dimerization. (C) Gel filtration to analyze the dimerization status of Emg1. Gel filtration was performed on a Superose 12 column with marker proteins of 25 (Chymotrypsinogen), 43 (Ovalbumin), 67 (Albumin), 158 kDa (Aldolase) and HisZZ-Emg1. An overlay of the elution profiles is shown. The fusion protein has a calculated molecular weight of ∼46 kDa for the monomer, but the large majority of it elutes between the 158 and the 67 kDa markers, showing that it forms a homodimer in solution.

Figure 5.

Mutations in residues flanking the hydrophobic cavity in Emg1 abolish SAM binding. Recombinant wild-type Emg1 and mutant proteins were immobilized and incubated with [³H] SAM. After washing, bound fractions were eluted and the recovery of [³H] SAM was determined by scintillation counting. The results were normalized to the control (matrix without Emg1). All Emg1 mutants analyzed had almost entirely lost the ability to bind SAM.

Figure 6.

Emg1 mutants can complement for growth upon depletion of endogenous Emg1. Growth complementation in a background where the genomic EMG1 gene is under control of the GAL1 promoter was tested with plasmid-derived wild-type or mutant Emg1, or empty plasmid. Three SAM-binding mutants (A–C) and the RNA-binding mutant R88D (D–F) were tested for growth complementation. Dilutions of cultures were spotted on plates containing galactose (A and D) or under depletion conditions on glucose (B and E). Growth of the strains was also analyzed in liquid culture (C and F). Both wild-type Emg1 as well as the RNA- and SAM-binding defective mutants complement the growth phenotype of Emg1 depletion, whereas cells containing only the empty plasmid show strong growth impairment.

Figure 7.

Mutations in the SAM-binding site of Emg1 do not affect ribosome biogenesis. As described in Figure 6, strains depleted of Emg1 were complemented with plasmid-derived wild-type or mutant Emg1, or with the empty vector (control). Total RNA was isolated before depletion (0 h) and after Emg1 depletion for 8, 16 and 24 h. RNA was separated on 1.2% agarose glyoxal gels (A) or 8% polyacrylamide/urea gels (B), and analyzed by northern hybridization. Probe numbers are shown on the left of the corresponding panels. rRNA intermediates and mature rRNAs are labeled on the right. In the control strain, depletion of Emg1 leads to strong ribosome biogenesis defects, whereas the strains complemented with wild-type Emg1 or the mutants do not show such effects.