Abstract of the PDB Structure's related Publication:
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.
Nep1 catalyzes the SAM-dependent methylation of a pseudouridine residue in the loop of hairpin 35 of 18S rRNA (position 1191 = position 966 in E.coli 16 rRNA, normally filled by m2G966 catalyzed by E. coli RsmD). Methylation occurs after the target U is first transformed into Y by a H/ACA RNP (snR35/Cbj5) pseudouridylation system. Yeast nucleolar Nep1 (formerly named Ermg1) is a homolog of Methanocaldococcus jannaschii Nep1 (Archaea). However, in naturally occurring 18S rRNA of Saccharomyces carsbergensis (and probably also S. cerevisiae), m1acp3Y is found, attesting that m1Y1191 is normally further hypermodified by an enzyme that has still to be identified. The crystal structure of Nep1 reveals a fold which is very similar to the conserved core fold of the SPOUT-class methyltransferases superfamily, including Nep1 of M. jannaschii. This highly conserved eukaryotic nucleolar protein Nep1 plays an essential role in 18S rRNA processing and ribosome biogenesis. It binds to a consensus 6-nt RNA-binding motif found in yeast 18S rRNA and facilitates the incorporation of ribosomal protein Rps19 during the formation of pre-ribosomes.