Recombinant Schizosaccharomyces pombe Nucleolar GTP-binding protein 2 (nog2), partial

What is Nog2 and what is its fundamental function in S. pombe?

Nog2 is an essential nuclear GTPase that is evolutionarily conserved between yeast and humans. In Schizosaccharomyces pombe, Nog2 plays a critical role during RNA rearrangements within the precursor large (pre-60S) subunit, functioning as a key checkpoint before nuclear export of the ribosomal subunit . Recent studies have demonstrated that Nog2 directly monitors the formation of specific rRNA modifications, particularly the 2′-O-methylation of G2922, which serves as a structural checkpoint in the eukaryotic ribosome assembly pathway .

The protein functions by binding to specific regions of ribosomal RNA, with particular importance placed on its interaction with helix 92 (H92) of the pre-60S subunit. This interaction is crucial for cell viability, as it helps ensure proper ribosome assembly and subsequent nuclear export .

How conserved is Nog2 across species?

Nog2 shows remarkable evolutionary conservation between yeast species like Schizosaccharomyces pombe and Saccharomyces cerevisiae, extending to human cells. This conservation reflects the fundamental importance of Nog2 in ribosome biogenesis across eukaryotic organisms . The functional domains related to GTPase activity and RNA binding show particular conservation, suggesting these regions are essential for the protein's role in ribosome assembly.

Mutagenic scanning experiments have identified specific amino acid residues that are highly intolerant to mutation, particularly those involved in the interaction with H92 and the recognition of the 2′-O-methylated G2922 base. This strong resistance to mutation in these regions further emphasizes their evolutionary importance .

How can researchers express and purify recombinant Nog2 from S. pombe?

For expressing recombinant S. pombe Nog2, researchers have successfully employed several strategies:

Expression System Design:

• PCR amplification of the NOG2 locus (~500 nt upstream of the NOG2 start codon to ~80 nt downstream of the NOG2 stop codon) from genomic DNA

• Modification of internal restriction sites (e.g., replacing BsaI site with synonymous coding sequence G444>A)

• Removal of the single intron within the NOG2 locus for bacterial expression

• Incorporation of tags for purification and detection (e.g., 3xFLAG-6xHis tag)

Strain Construction Methods:

• Generation of strains like AJY4427 (KanMX::pGAL1:SPB1 NOG2∆intron-3xFLAG-6xHis) via YTK-compatible Cas9 protocols

• Use of specific sgRNA targeting the intron within NOG2 (e.g., 5′-TTTGATTCTTTCCAACCAAG-3′)

• Co-transformation with Cas9-sgRNA plasmid and linearized repair template

For purification, affinity chromatography using the His tag has proven effective, followed by size exclusion chromatography to ensure high purity for structural and functional studies.

What is the molecular basis for Nog2's recognition of rRNA modifications?

The interaction between Nog2 and rRNA modifications, particularly the 2′-O-methylation of G2922, represents a critical checkpoint in ribosome assembly. Cryo-EM structural studies have revealed the molecular basis for this recognition:

• The 2′-O-methylated G2922 (Gm2922) is flipped from its canonical position by a 2′-endo pucker into a channel that gates the active site of Nog2 .

• The 2′-O-methyl group of Gm2922 is accommodated at the entrance to Nog2's active site, creating a specific binding pocket that recognizes this modification .

• Arg389 of Nog2, which strongly resists amino acid substitutions in mutagenesis studies, stabilizes Gm2922 through a cation-π interaction with the guanosine base .

• On the opposite side of the channel, Ser208 forms a hydrogen bond with the 5′-phosphate of Ψ2923, providing additional stabilization of the interaction. This residue was also highly resistant to mutation in functional studies .

This recognition mechanism ensures that only properly modified pre-60S subunits are allowed to progress through the assembly pathway, thereby serving as a quality control checkpoint for ribosome biogenesis.

How does Nog2 function change during nitrogen depletion conditions?

During nitrogen depletion in S. pombe, significant transcriptional changes occur that affect ribosome biogenesis pathways. Within 20 minutes after nitrogen removal, genes involved in ribosome biogenesis are down-regulated, while genes involved in pyrimidine salvage and nucleotide catabolism are up-regulated .

These changes in gene expression patterns likely affect Nog2 function in two primary ways:

• Altered substrate availability: The down-regulation of ribosome biogenesis genes likely reduces the number of pre-60S ribosomal subunits available for Nog2 to process.

• Modified regulatory context: The cellular response to nitrogen starvation involves nucleosome eviction and histone acetylation changes, particularly increases in H3AcK9 levels. These chromatin changes occur in both promoters and coding regions of up-regulated genes .

While Nog2 was not directly examined in the nitrogen depletion studies, the fundamental changes to ribosome biogenesis pathways under these conditions would inevitably impact Nog2's role in pre-60S subunit maturation and export. This represents an important area for future research to understand how Nog2 function adapts to stress conditions.

What mutagenesis approaches can reveal functional domains of Nog2?

Massively parallel mutagenic scanning has proven highly effective for identifying functionally important regions of Nog2. The methodology involves:

• Library Generation: Creating a comprehensive library of NOG2 codon variants using mutagenic PCR. This approach can achieve coverage of approximately 50% of possible amino acid substitutions. Key technical considerations include:

  • Adjustment of dNTP concentrations (0.2 mM dATP and dGTP; 1 mM dTTP and dCTP) to alleviate bias in mutation types

  • Addition of manganese chloride (500 μM) after initial amplification cycles to increase mutation rate

• Selection System: Introduction of the variant library into a strain where the endogenous NOG2 gene is under control of a regulatable promoter (e.g., glucose-repressible GAL1 promoter), allowing for selection of functional mutants

• Sequence Analysis: Deep sequencing and analysis of variants to identify residues that are intolerant to mutation

• Structural Mapping: Projection of mutation intolerance data onto the protein structure to identify functionally critical regions

This approach successfully identified several key regions of Nog2, including those involved in interaction with H92 and recognition of the 2′-O-methylated G2922. Particularly notable was the discovery of mutation-intolerant residues surrounding Gm2922, highlighting this interaction as crucial for Nog2 function .

How do single amino acid changes in Nog2 affect its ability to bypass Gm2922 methylation requirements?

Single amino acid changes in Nog2 can remarkably bypass the cellular dependence on Gm2922 methylation, indicating direct monitoring of this modification by Nog2. Two key substitutions have been identified:

• Thr195Arg substitution: This positively charged substitution is predicted to bind more strongly to the negatively charged H92 phosphodiester backbone. Expression of NOG2-T195R restored growth in the methyltransferase-deficient spb1-D52A strain to near wild-type levels .

• His392Arg substitution: This variant, identified through screening of randomly generated suppressors of spb1-D52A, also restored growth to near wild-type levels. Like T195R, this substitution is predicted to increase electrostatic binding with the phosphodiester backbone of H92 .

The expression of NOG2-H392R was shown to restore 60S biogenesis and nuclear export of the pre-60S in strains lacking 2′-O-methylated G2922 . This finding strongly suggests that Nog2 directly monitors the formation of Gm2922 to gate nuclear export of the large ribosomal subunit, and that increased binding affinity to H92 can compensate for the absence of this modification.

What approaches can be used to study Nog2's role in replication fork barriers?

Studies in S. pombe have established connections between nuclear proteins and replication fork barriers (RFBs). While Nog2 itself has not been directly implicated in RFB function, methodologies used to study these processes can be adapted to investigate potential roles of Nog2:

• Site-specific replication fork barrier systems: The RTS1 (Replication Termination Sequence 1) barrier system in S. pombe provides a valuable tool for investigating protein interactions with replication forks. This system could be adapted to study whether Nog2 plays any role in fork progression or restart .

• 2D gel analysis: Two-dimensional gel electrophoresis has been effectively used to analyze replication intermediates at barriers. This methodology can detect changes in fork arrest and restart efficiencies when specific factors are absent or mutated .

• Biotin proximity labeling: TurboID-based proximity labeling, followed by mass spectrometry analysis, can identify proteins within close proximity to Nog2 during various cellular processes, including potential roles during DNA replication .

• Genetic interaction studies: Systematic analysis of genetic interactions between Nog2 and factors known to be involved in replication fork progression or restart can provide insights into potential functional relationships.

How can researchers effectively study Nog2's role in checkpoint function during ribosome assembly?

To investigate Nog2's checkpoint function during ribosome assembly, several methodologies have proven effective:

• Genetic suppressor screens: Screening for suppressors of methylation-deficient phenotypes (e.g., spb1-D52A) to identify Nog2 variants that can bypass the requirement for specific rRNA modifications .

• Cryo-EM structural analysis: Solving structures of nascent 60S subunits from cells with unmethylated G2922 to directly visualize how the absence of methylation affects Nog2 engagement .

• Nuclear export assays: Monitoring the nuclear export of pre-60S subunits in various genetic backgrounds to assess how Nog2 mutations or rRNA modification defects impact this process .

• Ribosome assembly profiling: Using sucrose gradient centrifugation to analyze ribosome assembly intermediates and detect defects in 60S biogenesis associated with Nog2 mutations or rRNA modification defects .

These approaches can be integrated to provide comprehensive insights into how Nog2 functions as a structural checkpoint in ribosome assembly pathways.

What methodologies can effectively elucidate the GTPase activity of Nog2?

Although the search results don't provide specific details about measuring Nog2's GTPase activity, based on standard approaches for studying GTPases, researchers could employ:

• In vitro GTPase assays: Measuring GTP hydrolysis rates using purified recombinant Nog2 with radioactive [γ-32P]GTP or colorimetric/fluorometric assays that detect inorganic phosphate release.

• Structure-function analysis: Using the mutation data to identify key residues in the GTPase domain and testing how mutations affect GTP binding and hydrolysis.

• GTP-binding assays: Measuring the affinity of Nog2 for GTP using techniques like isothermal titration calorimetry (ITC) or fluorescence spectroscopy with fluorescent GTP analogs.

• Nucleotide exchange assays: Determining the rates of GDP release and GTP binding to understand the kinetics of Nog2's GTPase cycle.

These approaches would help characterize how Nog2's GTPase activity relates to its role in monitoring rRNA modifications and facilitating ribosome assembly.

How should researchers interpret confounding data when studying Nog2 function?

When encountering contradictory results in Nog2 functional studies, researchers should consider several possible explanations and analytical approaches:

• Strain background effects: Different S. pombe strains may have subtle genetic differences that affect Nog2 function or the ribosome assembly pathway. For example, previous studies of Rtf2 in RTS1 barrier function showed contrasting results depending on whether plasmid-based or genomic systems were used .

• Experimental system differences: Results from in vitro versus in vivo experiments, or from different expression systems, may yield apparently contradictory data. The search results show that previous plasmid-based studies of RTS1 region A suggested it was dependent on Rtf2, but genomic studies showed it was dispensable . Similar considerations may apply to Nog2 studies.

• Conditional dependencies: The function of Nog2 may be context-dependent, varying with growth conditions or cell cycle stage. For example, under nitrogen depletion, ribosome biogenesis genes are down-regulated in S. pombe , which could affect interpretation of Nog2 phenotypes.

• Technical approach limitations: Different techniques (genetic, biochemical, structural) have inherent limitations. For example, proximity labeling experiments with Nog2 did not identify replication-associated factors, contrasting with human RTF2 studies that showed enrichment at nascent chromatin . This may reflect differences in sensitivity between techniques rather than true biological differences.

What strategies can address challenges in purifying functional recombinant Nog2?

Although the search results don't directly address purification challenges, based on the information provided about Nog2's structure and function, researchers might consider these strategies:

• Optimizing expression constructs: Removing the intron from the NOG2 gene and using codon-optimized sequences as demonstrated in the search results .

• Expression condition screening: Testing various induction conditions (temperature, inducer concentration, duration) to maximize soluble protein yield.

• Solubility enhancement: Using solubility-enhancing tags (e.g., MBP, SUMO) or co-expressing with known binding partners from the pre-60S ribosomal subunit.

• Functional assay development: Developing robust assays to confirm that purified Nog2 retains GTPase activity and rRNA binding capability.

• Structural stabilization: Including GTP analogs or stabilizing mutations based on the mutagenesis data to increase protein stability during purification.

How might Nog2 function differ between model organisms and human cells?

While Nog2 is conserved between yeast and humans, functional differences may exist that could be important for translating basic research findings to human cell biology:

• Regulatory mechanisms: Humans likely have additional layers of regulation controlling Nog2 function that are absent in yeast, potentially including post-translational modifications or regulatory protein interactions.

• rRNA modification patterns: The specific pattern and prevalence of rRNA modifications may differ between species, potentially affecting how Nog2 monitors these modifications.

• Subcellular localization: The human ortholog may have more complex localization patterns or shuttling behavior between cellular compartments.

• Integration with stress responses: Human Nog2 may be integrated with stress response pathways in ways that differ from yeast, potentially affecting how ribosome biogenesis responds to cellular stresses.

Comparative studies between S. pombe, S. cerevisiae, and human cells could reveal conserved mechanisms and species-specific adaptations of Nog2 function in ribosome biogenesis.

How can researchers integrate Nog2 functional studies with broader ribosome biogenesis pathways?

To place Nog2 function in the broader context of ribosome biogenesis, researchers should consider:

• Temporal mapping of assembly factors: Determining the precise timing of Nog2 action relative to other assembly factors would help clarify its role in the sequential assembly pathway.

• Integration with other checkpoint mechanisms: Investigating how the Nog2-dependent checkpoint integrates with other quality control mechanisms in ribosome assembly.

• Connections to translation regulation: Exploring whether defects in Nog2 function lead to subtle alterations in ribosome function that affect translation fidelity or efficiency.

• Response to cellular stresses: Examining how various stresses (beyond nitrogen depletion) affect Nog2 function and the checkpoint it enforces.

These approaches would help develop a more comprehensive understanding of how Nog2 contributes to the highly regulated process of ribosome biogenesis.