Genetic interactions of yeast NEP1 (EMG1), encoding an essential factor in ribosome biogenesis

Abstract

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.

Yeast Rrp8p, a novel methyltransferase responsible for m1A 645 base modification of 25S rRNA

“…Consequently, we chose to mutate the equivalent residue and generated the substitution mutants, rrp8 – G209R and rrp8 – G209A . A serial dilution assay of the rrp8-ΔC mutant revealed the previously observed cold-sensitive phenotype and the hypersensitivity to paromomycin (28,29). The mutants expressing Rrp8–G209R and Rrp8–G209A also showed these phenotypes.…”

“…The accumulation of an aberrant 21S rRNA in the Δ rrp8 deletion strain illustrated the role of Rrp8 in affecting rRNA processing at site A2 (28). In our previous study, we have also shown that Rrp8 plays a modest but yet significant role in 40S subunit biogenesis (29). Bioinformatics studies using distant homology detection and fold recognition predicted Rrp8 to be a class I MTase (Rossmann-like fold) with the MTase domain located in the C-terminal part of the protein (26).…”

“…In our previous study, we made evident that the synthetic lethal interaction between a suppressed Δ nep1 and Δ rrp8 mutant is the result of defects in 40S biogenesis (29), which was the basis for our assumption of Rrp8 being a 40S MTase. Similarly, we next analysed another synthetic lethal interaction of the rrp8-ΔC mutant with a gar1 mutant.…”

“…This is in accordance to its involvement in processing at site A2 and the genetic interaction with the essential H/ACA snoRNP protein, Gar1 (28). We have also reported a genetic interaction of RRP8 with the essential biogenesis factor NEP1 , arguing for a specific role in 40S subunit biogenesis (29). Interestingly, Rrp8 was also found in early pre-60S complexes containing the snoRNA-independent MTase Spb1, suggesting that Rrp8 remains associated to the 27S A2 pre-rRNA directly after A2 cleavage (30).…”

Human diseases of the SSU processome

“…Studies in yeast show that EMG1 genetically interacts with Utp30, another component of the SSU processome. This interaction supports incorporation of the ribosomal protein Rps19 into the assembling pre-ribosome [46]. In addition, overexpression of Rps19 rescues growth defects in EMG1 deficient yeast [47].…”