Sequence-directed RNA remodeling within a topologically complex RNP substrate

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

doi:10.1101/2022.02.24.481786

Cited by 4 publications

(10 citation statements)

References 36 publications

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“…Figures were generated with UCSF Chimera 38 , Chimera X 39 , and the PyMOL Molecular Graphics System (Version 2.0 Schödinger, LLC.). 11 . b, detail of the interaction between the methyltransferase domain of Spb1 (teal cartoon) and helix 92 (orange).…”

Section: Cryo-em Model Building and Refinementmentioning

confidence: 99%

“…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 7,8,11 (Fig. 1a, 1b).…”

Section: Introductionmentioning

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.…”

Section: Introductionmentioning

confidence: 99%

“…Because the presence of Nog2-GDP did not cause any structural defects or instabilities in late nucleoplasmic intermediates, we instead hypothesized that the 60S biogenesis defect of the spb1 D52A strain arises during the nucleolar/nucleoplasmic transition of pre-60S particles. This transition is initiated by the removal of a set of RBFs by the AAA+ ATPase Rea1, triggering a rearrangement of the L1 stalk that allows it to dock onto the pre-60S core 11, 13 . We propose that this initial engagement relies on the interaction between Nog2 and h92 (Fig.…”

Section: Introductionmentioning

confidence: 99%

“…Figure 1 -Disruption of Spb1 methylation of G2922 results in premature Nog2 GTP hydrolysis. a, position of the methyltransferase Spb1 in the State E2 cryo-EM reconstruction11 . b, detail of the interaction between the methyltransferase domain of Spb1 (teal cartoon) and helix 92 (orange).…”

mentioning

confidence: 99%

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

Sekulski

Cruz

Weirich

et al. 2022

Preprint

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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 shows the direct involvement of unmodified G2922 in Nog2 GTPase activation. Intragenic and dosage suppressors indicate that the biogenesis defect in the spb1D52A strain is caused by reduced initial recruitment of Nog2 to early nucleoplasmic 60S intermediates. We propose that G2922 methylation levels regulate Nog2 stability on the pre-60S near the nucleolar/nucleoplasmic phase boundary, forming a kinetic checkpoint to regulate 60S production. Because multiple K-loop GTPases are involved in the assembly of ribosomes and other RNPs, our findings provide a template to study the role of RNA trans factors in modulating the regulatory functions of this important family of enzymes.

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Section: Cryo-em Model Building and Refinementmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

mentioning

confidence: 99%

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

Sekulski

Cruz

Weirich

et al. 2022

Preprint

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Tuning Strand Displacement Kinetics Enables Programmable ZTP Riboswitch Dynamic Rangein vivo

Bushhouse

Lucks

2022

Preprint

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Recent work has shown that transcriptional riboswitches function through internal strand displacement mechanisms that guide the formation of alternative structures which drive regulatory outcomes. Here we sought to investigate this phenomenon using theClostridium beijerinckii pflZTP riboswitch as a model system. Using functional mutagenesis within vivogene expression assays inE. coli, we show that mutations designed to slow strand displacement of the expression platform enable precise tuning of riboswitch dynamic range (2.4–34-fold), depending on the type of kinetic barrier introduced, and the position of the barrier relative to the strand displacement nucleation site. We also show that expression platforms from a range of differentClostridiumZTP riboswitches contain sequences that impose these barriers to affect dynamic range in these different contexts. Finally, we use sequence design to flip the regulatory logic of the riboswitch to create a transcriptional OFF-switch, and show that the same barriers to strand displacement tune dynamic range in this synthetic context. Together, our findings further elucidate how strand displacement can be manipulated to alter the riboswitch decision landscape, suggesting that this could be a mechanism by which evolution tunes riboswitch sequence, and providing an approach to optimize synthetic riboswitches for biotechnology applications.

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Principles of human pre-60Sbiogenesis

Broeck

Klinge

2023

Preprint

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During early stages of human large ribosomal subunit (60S) biogenesis, an ensemble of assembly factors establishes and fine-tunes the essential RNA functional centers of pre-60Sparticles by an unknown mechanism. Here, we report a series of cryo-electron microscopy structures of human nucleolar and nuclear pre-60Sassembly intermediates at resolutions of 2.5-3.2 angstroms. These structures show how protein interaction hubs tether assembly factor complexes to nucleolar particles and how GTPases and ATPases couple irreversible nucleotide hydrolysis steps to the installation of functional centers. Nuclear stages highlight how a conserved RNA processing complex, the rixosome, couples large-scale RNA conformational changes to pre-rRNA processing by the RNA degradation machinery. Our ensemble of human pre-60Sparticles provides a rich foundation to elucidate the molecular principles of ribosome formation.

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Sequence-directed RNA remodeling within a topologically complex RNP substrate

Cited by 4 publications

References 36 publications

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

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

Tuning Strand Displacement Kinetics Enables Programmable ZTP Riboswitch Dynamic Rangein vivo

Principles of human pre-60Sbiogenesis

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