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Structure paper

TitlerRNA methylation by Spb1 regulates the GTPase activity of Nog2 during 60S ribosomal subunit assembly.
Journal, issue, pagesNat Commun, Vol. 14, Issue 1, Page 1207, Year 2023
Publish dateMar 2, 2023
AuthorsKamil Sekulski / Victor Emmanuel Cruz / Christine S Weirich / Jan P Erzberger /
PubMed AbstractBiogenesis 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 ...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 (spb1) 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-AlF 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.
External linksNat Commun / PubMed:36864048 / PubMed Central
MethodsEM (single particle)
Resolution2.34 - 2.9 Å
Structure data

26651

7uoo

EMDB-26651, PDB-7uoo:
Nucleoplasmic pre-60S intermediate of the Nog2 containing pre-rotation state
Method: EM (single particle) / Resolution: 2.34 Å

26686

7uqb

EMDB-26686, PDB-7uqb:
Nucleoplasmic pre-60S intermediate of the Nog2 containing pre-rotation state from a SPB1-D52A strain with AlF4
Method: EM (single particle) / Resolution: 2.43 Å

EMDB-26689: Locally refined 5S rRNP map from the nucleoplasmic pre-60S intermediate of the Nog2 containing pre-rotation state
Method: EM (single particle) / Resolution: 2.63 Å

26703

7uqz

EMDB-26703, PDB-7uqz:
Nucleoplasmic pre-60S intermediate of the Nog2 containing pre-rotation state from a SPB1 D52A strain
Method: EM (single particle) / Resolution: 2.44 Å

26799

7uui

EMDB-26799, PDB-7uui:
Nucleoplasmic pre-60S intermediate of the Nog2 containing post-rotation state from a SPB1 D52A strain
Method: EM (single particle) / Resolution: 2.9 Å

26941

7v08

EMDB-26941, PDB-7v08:
Nucleoplasmic pre-60S intermediate of the Nog2 containing pre-rotation state from a Spb1 D52A suppressor 3 strain
Method: EM (single particle) / Resolution: 2.36 Å

Chemicals

ChemComp-MG:
Unknown entry

ChemComp-B3P:
2-[3-(2-HYDROXY-1,1-DIHYDROXYMETHYL-ETHYLAMINO)-PROPYLAMINO]-2-HYDROXYMETHYL-PROPANE-1,3-DIOL / pH buffer*YM / Bis-tris propane

ChemComp-ZN:
Unknown entry

ChemComp-GDP:
GUANOSINE-5'-DIPHOSPHATE / GDP, energy-carrying molecule*YM / Guanosine diphosphate

ChemComp-GTP:
GUANOSINE-5'-TRIPHOSPHATE / GTP, energy-carrying molecule*YM / Guanosine triphosphate

ChemComp-K:
Unknown entry

ChemComp-HOH:
WATER / Water

ChemComp-ALF:
TETRAFLUOROALUMINATE ION

Source
  • saccharomyces cerevisiae by4741 (yeast)
KeywordsRIBOSOME / ribosome biogenesis / K-loop GTPase / GTPase / GTPase transition state / GDP aluminum fluoride / 5S rRNP

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