A single 2′-O-methylation of ribosomal RNA gates assembly of a functional ribosome

Abstract

RNA modifications are widespread in biology and abundant in ribosomal RNA. However, the importance of these modifications is not well understood. We show that methylation of a single nucleotide, in the catalytic center of the large subunit, gates ribosome assembly. Massively parallel mutational scanning of the essential nuclear GTPase Nog2 identified important interactions with rRNA, particularly with the 2′-O-methylated A-site base Gm2922. We found that methylation of G2922 is needed for assembly and efficient nuclear export of the large subunit. Critically, we identified single amino acid changes in Nog2 that completely bypass dependence on G2922 methylation and used cryoelectron microscopy to directly visualize how methylation flips Gm2922 into the active site channel of Nog2. This work demonstrates that a single RNA modification is a critical checkpoint in ribosome biogenesis, suggesting that such modifications can play an important role in regulation and assembly of macromolecular machines.

rRNA methylation by Spb1 regulates the GTPase activity of Nog2 during 60S ribosomal subunit assembly

“…5d). Our structural, genetic, and localization data are in agreement with a recently published study on the function of G2922 methylation during 60S assembly 29 .…”

Oleaginous Heterotrophic Dinoflagellates—Crypthecodiniaceae

“…The truncation-insertion events ( Figure 2 ) would not be fully reflected on a phylogenetic tree with fixed alignment gaps allowances of most parsimonious algorithms. ITS processing is involved in the maturation of 60S pre-ribosomes prior to nuclear export [ 44 ], which will be crucial in dinoflagellates, as there is no nuclear envelope breakdown. The changes in ITS1 also co-occurred with changes at ITS2, strongly indicating co-evolved functional significance.…”

Quality control ensures fidelity in ribosome assembly and cellular health

“…During nuclear pre-60S assembly in yeast, the methyltransferase Spb1 methylates G2922 of 25S rRNA at its 2′-OH ( Lapeyre and Purushothaman, 2004 ; Hansen et al, 2002 ). The successful installation of this modification in the A-site is then reinspected in a subsequent step, the binding and activation of the GTPase Nog2 ( Cruz et al, 2022 ; Yelland et al, 2023 ). While modified G m 2922 engages the Nog2 active site stably ( Cruz et al, 2022 ; Yelland et al, 2023 ), it does not allow for GTPase activation as the methylation blocks an essential hydrogen bonding network to the γ-phosphate, present with unmethylated G2922 ( Cruz et al, 2022 ).…”

“…The successful installation of this modification in the A-site is then reinspected in a subsequent step, the binding and activation of the GTPase Nog2 ( Cruz et al, 2022 ; Yelland et al, 2023 ). While modified G m 2922 engages the Nog2 active site stably ( Cruz et al, 2022 ; Yelland et al, 2023 ), it does not allow for GTPase activation as the methylation blocks an essential hydrogen bonding network to the γ-phosphate, present with unmethylated G2922 ( Cruz et al, 2022 ). Thus, G2922 leads to rapid GTP hydrolysis and thus inactivation of Nog2, which appears to mostly dissociate.…”

“…Thus, the Nog2 GTPase activity is used as a timer, which retroactively inspects G2922 methylation in a manner that allows the mistake to be fixed, as Nog2 dissociation also allows Spb1 the chance to rebind and methylate the RNA. While bypass mutations for this proofreading checkpoint have been identified ( Cruz et al, 2022 ; Yelland et al, 2023 ), it remains unknown whether these produce misassembled and dysfunctional ribosomes, thus precluding us from unambiguously labeling this a QC step.…”