TY - JOUR
T1 - rRNA methylation by Spb1 regulates the GTPase activity of Nog2 during 60S ribosomal subunit assembly
AU - Sekulski, Kamil
AU - Cruz, Victor Emmanuel
AU - Weirich, Christine S.
AU - Erzberger, Jan P.
N1 - Funding Information:
We thank Daniel Stoddard and Jose Martinez at the UTSW Cryo-Electron Microscopy Facility, funded in part by the CPRIT Core Facility Support Award RP170644, James Chen at the UTSW Structural Biology Lab and Matthew Parker for help with light microscopy. A portion of this research was supported by NIH grant U24GM129547 and performed at the PNCC at OHSU and accessed through EMSL (grid.436923.9), a DOE Office of Science User Facility sponsored by the Office of Biological and Environmental Research, with the assistance of Theo Humphreys. We thank V. Panse for sharing plasmids pRS316-uL18-GFP and pRS316-uS5-GFP. The authors would also like to acknowledge the Quantitative Light Microscopy Core, a shared resource of the Harold C. Simmons Cancer Center, supported in part by an NCI Cancer Center Support Grant, 1P30 CA142543-01 and the UTSW Proteomics Core. J.P.E. was supported by the Cancer Prevention and Research Institute of Texas (RR150074), the Welch Foundation (I-1897), the UTSW Endowed Scholars Fund and the National Institutes of Health (GM135617-01).
Publisher Copyright:
© 2023, The Author(s).
PY - 2023/12
Y1 - 2023/12
N2 - 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.
AB - 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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U2 - 10.1038/s41467-023-36867-5
DO - 10.1038/s41467-023-36867-5
M3 - Article
C2 - 36864048
AN - SCOPUS:85149328843
SN - 2041-1723
VL - 14
JO - Nature communications
JF - Nature communications
IS - 1
M1 - 1207
ER -