The BowenConradi syndrome protein Nep1 (Emg1) has a dual role in eukaryotic ribosome biogenesis, as an essential assembly factor and in the methylation of 1191 in yeast 18S rRNA

Author： Meyer Britta

Publisher： Oxford University Press

ISSN： 1362-4962

Source： Nucleic Acids Research, Vol.39, Iss.4, 2011-03, pp. : 1526-1537

Disclaimer: Any content in publications that violate the sovereignty, the constitution or regulations of the PRC is not accepted or approved by CNPIEC.

Previous Menu Next

Abstract

The Nep1 (Emg1) SPOUT-class methyltransferase is an essential ribosome assembly factor and the human BowenConradi syndrome (BCS) is caused by a specific Nep1^D86G 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 (m1acp3). Specific ¹⁴C-methionine labelling of 18S rRNA in yeast mutants showed that Nep1 is not required for acp-modification but suggested a function in 1191 methylation. ESI MS analysis of acp-modified -nucleosides in a nep1-mutant showed that Nep1 catalyzes the 1191 methylation in vivo. Remarkably, the restored growth of a nep1-1^ts mutant upon addition of S-adenosylmethionine was even observed after preventing U1191 methylation in a snr35 mutant. This strongly suggests a dual Nep1 function, as 1191-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.