Recombinant Ashbya gossypii ATP-dependent rRNA helicase SPB4 (SPB4), partial

What is Ashbya gossypii ATP-dependent rRNA helicase SPB4?

SPB4 is an essential ATP-dependent RNA helicase found in the filamentous fungus Ashbya gossypii. It belongs to the DEAD-box family of helicases characterized by conserved motifs including the Walker B (DEAD) motif. The protein functions in ribosome biogenesis, specifically in the assembly of 60S ribosomal subunits, where it plays a critical role in rRNA remodeling during pre-60S particle maturation . The recombinant partial form is commonly used for research purposes and typically has a purity of >85% as determined by SDS-PAGE .

How does SPB4 compare to homologous proteins in other organisms?

SPB4 in A. gossypii shares significant homology with Spb4p in Saccharomyces cerevisiae. This evolutionary conservation is consistent with the extensive synteny (>90%) observed between A. gossypii and S. cerevisiae genomes . In S. cerevisiae, Spb4p depletion results in underaccumulation of 60S ribosomal subunits and inhibition of 27SB pre-rRNA processing, leading to reduced synthesis of 25S/5.8S rRNAs . This functional conservation makes A. gossypii SPB4 a valuable comparative model for studying evolutionary aspects of ribosome assembly mechanisms.

What is the biological context of A. gossypii as a model organism?

A. gossypii is a filamentous fungus that naturally grows as multinucleated hyphae and is a cotton pathogen transmitted by insects. It has gained significance as a model organism due to:

• Its extensive synteny with the S. cerevisiae genome

• Its amenability to genetic manipulation through homologous recombination

• Its commercial importance as a natural overproducer of riboflavin (vitamin B2)

These characteristics make A. gossypii and its proteins, including SPB4, valuable for both basic research and biotechnological applications.

What is known about the structural organization of SPB4?

SPB4 contains the characteristic structural elements of DEAD-box RNA helicases:

• Two RecA-like domains that form the catalytic core

• A nucleotide-binding pocket between the domains

• RNA-binding motifs

• A C-terminal domain (CTD) that interacts with rRNA

High-resolution cryo-EM studies (2.7 Å resolution) have revealed that SPB4 adopts a closed conformation when bound to its pre-ribosomal substrate, with the two RecA domains tightly associated even after ATP hydrolysis .

How does SPB4 interact with its rRNA substrate during ribosome biogenesis?

SPB4 specifically interacts with the H62/H63/H63a region of rRNA during pre-60S ribosome assembly. Structural analysis has shown that:

• SPB4 binds to this region and maintains it in an "immature alternate conformation"

• The helicase's RecA domains clamp around the RNA substrate

• The CTD of SPB4 forms tight interactions with rRNA, potentially locking the catalytic core in its closed state

• This interaction facilitates later rRNA restructuring essential for proper 60S subunit maturation

These interactions differ from the canonical model of DEAD-box helicase action, as SPB4 remains bound to its RNA substrate in a closed conformation even after ATP hydrolysis.

What is the catalytic mechanism of SPB4 and how does it differ from canonical DEAD-box helicases?

SPB4 exhibits an unusual catalytic mechanism compared to other DEAD-box helicases:

This atypical behavior allows SPB4 to stabilize the restructured rRNA in an immature conformation until subsequent assembly factors trigger its release, ensuring proper timing of ribosomal maturation events .

How should researchers handle recombinant A. gossypii SPB4?

For optimal handling of recombinant SPB4:

• Storage: Store lyophilized form at -20°C/-80°C (shelf life: 12 months); liquid form at -20°C/-80°C (shelf life: 6 months)

• Reconstitution: Briefly centrifuge vial before opening; reconstitute in deionized sterile water to 0.1-1.0 mg/mL

• Stability: Add glycerol to 5-50% final concentration (50% recommended) and aliquot for long-term storage

• Working aliquots: Store at 4°C for up to one week

• Freeze-thaw cycles: Avoid repeated freezing and thawing

What expression systems are suitable for producing recombinant SPB4?

Based on available information, E. coli is a suitable expression system for producing recombinant A. gossypii SPB4 . For successful expression:

• Optimize codon usage for E. coli if necessary

• Consider using a tag system for purification (tag type may vary based on manufacturing process)

• Express as a partial protein if full-length expression presents challenges

• Aim for >85% purity as verified by SDS-PAGE

• Verify RNA-stimulated ATPase activity, which can be abolished by a point mutation in the catalytic DEAD (Walker B) motif

What techniques are most effective for studying SPB4's interactions with pre-ribosomal complexes?

These approaches have successfully revealed SPB4's structure in complex with pre-ribosomal particles and identified its rRNA restructuring target in the H62/H63/H63a region .

How can researchers differentiate between the functions of SPB4 and other helicases in A. gossypii?

To differentiate between the functions of SPB4 and other helicases:

• Genetic approaches: Create conditional mutants or use depletion systems for each helicase and analyze specific defects in ribosome biogenesis

• Biochemical specificity: Determine the specific rRNA regions targeted by each helicase using CRAC (crosslinking and analysis of cDNA) or similar techniques

• Structural comparisons: Use cryo-EM to analyze structural differences in how each helicase interacts with pre-ribosomal particles

• Protein interaction networks: Identify unique binding partners for each helicase using co-immunoprecipitation or proximity labeling approaches

• Temporal dynamics: Analyze the timing of association and dissociation of each helicase during ribosome assembly using time-resolved approaches

What is the significance of SPB4's post-catalytic ADP-bound state?

The observation that SPB4 remains in a closed conformation with ADP bound after ATP hydrolysis represents a significant deviation from the canonical model of DEAD-box helicase function. This finding has important implications:

• It challenges existing models of DEAD-box helicase mechanisms that suggest ATP hydrolysis triggers immediate domain reopening and substrate release

• It indicates that on complex substrates like pre-ribosomes, additional factors may influence helicase conformational states

• It suggests SPB4's function involves not just ATP-dependent RNA unwinding but also stabilization of alternative RNA conformations

• It implies a coordinated release mechanism where additional assembly factors trigger SPB4 dissociation at the appropriate time

• It may represent a specialized adaptation for precise temporal control of ribosome assembly

This mechanism appears to "set the stage for the correct formation of the H61-H62-H63-H64 rRNA region as an important prerequisite for the further maturation of rRNA domain IV" .

How might researchers investigate the interactions between SPB4 and other pre-60S assembly factors?

To study interactions between SPB4 and other pre-60S assembly factors:

• Proximity-based proteomics: Use BioID or APEX labeling with SPB4 as bait to identify proteins in close proximity during ribosome assembly

• Co-immunoprecipitation with staged purification: Isolate complexes at different maturation stages to map the temporal dynamics of factor association

• Structural studies: Perform cryo-EM on particles isolated using different baits to visualize the arrangement of multiple factors simultaneously

• Genetic interaction screens: Identify synthetic lethality or suppression between mutations in SPB4 and other assembly factors

• In vitro reconstitution: Attempt stepwise assembly of minimal complexes to determine direct interactions and functional interdependencies

These approaches would help elucidate how SPB4 coordinates with factors like Rrp17, Noc2, Loc1, Noc3, and Spb1 during pre-60S maturation .

What are common challenges in working with recombinant SPB4 and how can they be addressed?

Researchers working with recombinant SPB4 may encounter several challenges:

Careful attention to these factors can improve experimental outcomes when working with recombinant SPB4 .

How can researchers effectively study SPB4 function in the context of A. gossypii as a model organism?

A. gossypii offers unique advantages as a model organism for studying SPB4 function:

• Genetic tools: A wide range of molecular tools is available for genetic manipulation of A. gossypii

• Promoter systems: Several characterized promoters are available for controlled expression, including strong promoters (PCCW12, PSED1) and medium/weak promoters (PTSA1, PHSP26, PAGL366C, etc.)

• Marker systems: The loxP-kanMX-loxP marker system allows marker recycling through Cre recombinase expression

• Fluorescent tagging: Expression cassettes for luciferase reporters and other fluorescent proteins enable in vivo monitoring

• Growth conditions: A. gossypii can be cultured on low-cost media, facilitating large-scale experiments

These tools enable sophisticated genetic and cell biological approaches to study SPB4 function in its native context.

What controls should be included in functional assays of SPB4?

For rigorous functional assays of SPB4, researchers should include:

• Catalytic mutant: A mutant in the Walker B (DEAD) motif, which abolishes RNA-stimulated ATPase activity, serves as a negative control

• ATP analogs: Non-hydrolyzable ATP analogs to distinguish between ATP binding and hydrolysis requirements

• Substrate controls: Both specific (authentic rRNA fragments) and non-specific RNA substrates to determine substrate specificity

• Reaction conditions: Optimization of salt, pH, and divalent cation concentrations to ensure optimal activity

• Time course analysis: Monitoring ATP hydrolysis and RNA unwinding over time to determine reaction kinetics

These controls help establish the specificity and mechanism of SPB4's helicase activity and its role in ribosome biogenesis.

What are important unanswered questions about SPB4 that warrant further investigation?

Several aspects of SPB4 function remain to be fully elucidated:

• The precise mechanism triggering SPB4 release from pre-ribosomal particles after ATP hydrolysis

• The structural basis for SPB4's unusual post-catalytic state

• The complete network of interactions between SPB4 and other assembly factors

• The evolutionary conservation of SPB4's mechanism across different fungal species

• The potential for targeting SPB4 or its interaction partners in antifungal drug development

Addressing these questions would advance our understanding of both ribosome assembly mechanisms and the specialized adaptations of DEAD-box helicases.

How might comparative studies between A. gossypii and S. cerevisiae inform our understanding of SPB4?

Comparative studies between these organisms could provide valuable insights:

• Analysis of SPB4/Spb4p complexes from both organisms using cryo-EM could reveal conserved and divergent structural features

• Complementation experiments testing whether A. gossypii SPB4 can rescue S. cerevisiae spb4 mutants would reveal functional conservation

• Evolution of regulatory networks controlling SPB4/Spb4p expression could illuminate adaptation to different growth patterns

• Differences in pre-rRNA processing pathways between the filamentous A. gossypii and unicellular S. cerevisiae could explain specialized roles of SPB4

• The interaction of SPB4 with species-specific assembly factors could reveal adaptations to different ecological niches

Such comparative approaches leverage the synteny between these genomes while exploring adaptations to different fungal lifestyles.