The Nep1 (Emg1) SPOUT-class methyltransferase is an essential ribosome assembly factor and the human Bowen–Conradi syndrome (BCS) is caused by a specific Nep1D86G mutation. We recently showed in vitro that Methanocaldococcus jannaschii Nep1 is a sequence-specific pseudouridine-N1-methyltransferase. Here, we show that in yeast the in vivo target site for Nep1-catalyzed methylation is located within loop 35 of the 18S rRNA that contains the unique hypermodification of U1191 to 1-methyl-3-(3-amino-3-carboxypropyl)-pseudouri-dine (m1acp3Ψ). Specific 14C-methionine labelling of 18S rRNA in yeast mutants showed that Nep1 is not required for acp-modification but suggested a function in Ψ1191 methylation. ESI MS analysis of acp-modified Ψ-nucleosides in a Δnep1-mutant showed that Nep1 catalyzes the Ψ1191 methylation in vivo. Remarkably, the restored growth of a nep1-1ts mutant upon addition of S-adenosylmethionine was even observed after preventing U1191 methylation in a Δsnr35 mutant. This strongly suggests a dual Nep1 function, as Ψ1191-methyltransferase and ribosome assembly factor. Interestingly, the Nep1 methyltransferase activity is not affected upon introduction of the BCS mutation. Instead, the mutated protein shows enhanced dimerization propensity and increased affinity for its RNA-target in vitro. Furthermore, the BCS mutation prevents nucleolar accumulation of Nep1, which could be the reason for reduced growth in yeast and the Bowen-Conradi syndrome.

Nep1 methylates the hypermodified c1191 base of 18S rRNA and has an additional essential function during ribosome biogenesis. It is strongly conserved in eukaryotes and a point mutation causes the human Bowen-Conradi syndrome. To identify Δnep1-specific genetic interactions, viable deletions were screened genome-wide (SGA). Due to its essential function, we used, for the first time, query strain (Δnep1) with two additive suppressor conditions (mcRPS19B, nop6-1). Nep1 interacting genes correspond to ribosome biogenesis (RPS18A, RPS18B, RRP8, EFG1, UTP30), to ribosome quality control (UBP3, BRE5, UBP6) and to ribosome functional control (DOM34, no-go decay). Deletions in ribosome quality and functional control genes were synthetically sick with Δnep1. They cope with malfunctions and the respective deletions strengthen the Δnep1 growth deficiency. Except for Δrps18b, deletions in the identified ribosome biogenesis genes were synthetically lethal with Δnep1. While the synthetic lethalities of Δrrp8 and Δefg1 may result from additive defects, the Δutp30 deletion seems to be in close functional relationship. The Δutp30 deletion itself has no phenotype but it enforced all nep1-1 ts mutant phenotypes. Furthermore, its overexpression partially restored the nep1-1 ts growth deficiency. Our genetic and biochemical data suggest that Utp30 and Nep1 act together during pre-ribosomal complex formation and, along with Rps18, provide the surface for the Rps19 assembly to the 90S pre-ribosome.