Sequence-specific remodeling of a topologically complex RNP substrate by Spb4

Cruz, Victor Emmanuel; Sekulski, Kamil; Peddada, Nagesh; Sailer, Carolin; Balasubramanian, Sahana; Weirich, Christine S.; Stengel, Florian; Erzberger, J.P.

doi:10.1038/s41594-022-00874-9

Cited by 19 publications

(49 citation statements)

References 68 publications

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“…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 ).…”

Section: Ribosome Assembly At a Glancementioning

confidence: 99%

“…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.…”

Section: Ribosome Assembly At a Glancementioning

confidence: 99%

“…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.…”

Section: Ribosome Assembly At a Glancementioning

confidence: 99%

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Quality control ensures fidelity in ribosome assembly and cellular health

Parker

Karbstein

2023

Journal of Cell Biology

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The coordinated integration of ribosomal RNA and protein into two functional ribosomal subunits is safeguarded by quality control checkpoints that ensure ribosomes are correctly assembled and functional before they engage in translation. Quality control is critical in maintaining the integrity of ribosomes and necessary to support healthy cell growth and prevent diseases associated with mistakes in ribosome assembly. Its importance is demonstrated by the finding that bypassing quality control leads to misassembled, malfunctioning ribosomes with altered translation fidelity, which change gene expression and disrupt protein homeostasis. In this review, we outline our understanding of quality control within ribosome synthesis and how failure to enforce quality control contributes to human disease. We first provide a definition of quality control to guide our investigation, briefly present the main assembly steps, and then examine stages of assembly that test ribosome function, establish a pass–fail system to evaluate these functions, and contribute to altered ribosome performance when bypassed, and are thus considered “quality control.”

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Section: Ribosome Assembly At a Glancementioning

confidence: 99%

Section: Ribosome Assembly At a Glancementioning

confidence: 99%

Section: Ribosome Assembly At a Glancementioning

confidence: 99%

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Quality control ensures fidelity in ribosome assembly and cellular health

Parker

Karbstein

2023

Journal of Cell Biology

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“…One such late modification is the 2´-OH methylation of G2922 (mG2922) within the A-site loop of rRNA helix 92 (H92) by the S-adenosyl-methionine (SAM)-dependent methyltransferase Spb1 (Fig. 1a, b) 7,8,11 . Notably, mG2922 is the only eukaryotic ribose methylation carried out in a snRNP-independent manner by a dedicated enzyme.…”

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confidence: 99%

“…Notably, mG2922 is the only eukaryotic ribose methylation carried out in a snRNP-independent manner by a dedicated enzyme. Spb1 is an essential structural component of nucleolar pre-60S intermediates 11,12 and strains carrying a mutation (spb1 D52A ) that abolishes G2922 methylation activity display a severe growth defect and impaired 60S biogenesis 7,8 . After Spb1 release and L1 stalk rearrangement 13 , the K + -dependent GTPase Nog2 engages the A-loop during nucleoplasmic 60S maturation steps (Fig.…”

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confidence: 99%

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

et al. 2023

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Biogenesis of the large ribosomal (60S) subunit involves the assembly of three rRNAs and 46 proteins, a process requiring approximately 70 ribosome biogenesis factors (RBFs) that bind and release the pre-60S at specific steps along the assembly pathway. The methyltransferase Spb1 and the K-loop GTPase Nog2 are essential RBFs that engage the rRNA A-loop during sequential steps in 60S maturation. Spb1 methylates the A-loop nucleotide G2922 and a catalytically deficient mutant strain (spb1D52A) has a severe 60S biogenesis defect. However, the assembly function of this modification is currently unknown. Here, we present cryo-EM reconstructions that reveal that unmethylated G2922 leads to the premature activation of Nog2 GTPase activity and capture a Nog2-GDP-AlF4− transition state structure that implicates the direct involvement of unmodified G2922 in Nog2 GTPase activation. Genetic suppressors and in vivo imaging indicate that premature GTP hydrolysis prevents the efficient binding of Nog2 to early nucleoplasmic 60S intermediates. We propose that G2922 methylation levels regulate Nog2 recruitment to the pre-60S near the nucleolar/nucleoplasmic phase boundary, forming a kinetic checkpoint to regulate 60S production. Our approach and findings provide a template to study the GTPase cycles and regulatory factor interactions of the other K-loop GTPases involved in ribosome assembly.

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