Recombinant Debaryomyces hansenii H/ACA ribonucleoprotein complex subunit 3 (NOP10)

What is the biological role of NOP10 in H/ACA ribonucleoprotein complexes?

NOP10 functions as an essential structural component of H/ACA ribonucleoprotein (RNP) complexes, which are responsible for pseudouridylation of RNAs. Within these complexes, NOP10 works cooperatively with other core proteins - Nhp2, Cbf5, and Gar1 (collectively referred to as WNCG) - to facilitate RNA binding and catalytic activity. The presence of NOP10 is critical for the proper assembly and function of these complexes, as demonstrated by the significant impact on complex formation when NOP10 is omitted .

In functional studies, RNP complexes formed with the complete set of proteins (including NOP10) show high enzymatic activity in pseudouridylation reactions. The interaction between NOP10 and other proteins, particularly Cbf5 and Nhp2, appears to create a structural backbone that stabilizes the RNP complex and positions the RNA substrate correctly within the pseudouridylation pocket .

How does Debaryomyces hansenii's halotolerant nature potentially affect NOP10 function?

Debaryomyces hansenii is an extremely halotolerant yeast capable of growing in media containing up to 25% NaCl or 18% glycerol . This remarkable adaptation to high-salt environments suggests that its cellular machinery, including RNA modification complexes containing NOP10, must remain functional under osmotic stress conditions.

The structural stability of H/ACA RNP complexes in D. hansenii may be enhanced compared to those in non-halotolerant organisms. D. hansenii maintains osmobalance through mechanisms involving sodium and potassium ions , which likely influence the ionic environment in which NOP10-containing complexes operate. Research examining NOP10 function under varying salt concentrations would provide valuable insights into how this protein maintains structural integrity and catalytic activity in extreme environments, potentially revealing adaptation-specific modifications in the protein sequence or structure.

What is known about the relationship between NOP10 and telomere maintenance?

In vitro experiments have provided evidence supporting a mechanism where NOP10 overexpression facilitates telomerase-dependent telomere length maintenance . While this relationship has been primarily studied in other contexts, it suggests a potential dual function for NOP10 in both RNA modification and telomere biology.

The H/ACA RNP complex containing NOP10 may play a role in processing or modifying the RNA component of telomerase. This connection between NOP10 and telomere maintenance suggests that alterations in NOP10 expression or function could potentially impact cellular aging and replicative capacity in D. hansenii, especially under stress conditions where maintaining genomic stability becomes crucial for survival.

What are the optimal methods for reconstituting active D. hansenii H/ACA RNP complexes containing NOP10?

Reconstitution of active H/ACA RNP complexes requires a stepwise assembly approach with purified recombinant proteins. Based on established protocols, the following methodology is recommended:

• Express and purify individual components (Nhp2, Nop10, Cbf5, and Gar1) using affinity chromatography

• Combine the proteins with in vitro transcribed RNA hairpins (such as the 5' hairpin H5 or 3' hairpin H3)

• Allow the complex to assemble under controlled conditions (typically 30°C in an appropriate buffer system)

• Verify complex formation through gel mobility shift assays or fluorescence-based techniques

For optimal activity, all four proteins (WNCG) should be incorporated, as research has shown that omission of any component significantly reduces catalytic function. The complete reconstitution typically results in a conformational change of the RNA hairpin structure that can be monitored using single-molecule FRET techniques, with the fully assembled complex showing distinct FRET signatures compared to partially assembled or unassembled components .

How can single-molecule FRET be utilized to study NOP10's role in complex assembly?

Single-molecule FRET (smFRET) provides valuable insights into the conformational changes that occur during H/ACA RNP assembly. The following methodology has proven effective:

• Design RNA hairpins with strategically placed FRET pairs (donor and acceptor fluorophores) across the pseudouridylation pocket

• Immobilize the labeled RNA using a biotin handle at the 3' end

• Monitor FRET efficiency changes during stepwise addition of proteins

• Analyze population distributions to identify distinct conformational states

In studies with hairpin RNAs like H5, two distinct FRET populations were observed: a low-FRET state (EFRET = 0.40) and an intermediate-FRET state (EFRET = 0.56-0.63). The shift from intermediate-FRET to low-FRET was dramatically enhanced (from 28% to 85%) when all four proteins, including NOP10, were present, indicating a cooperative action between NOP10 and other components that drives a conformational change in the RNA .

For determining protein positions within the complex, complementary experiments can be performed using labeled NOP10 (e.g., with fluorophores at specific lysine residues) and unlabeled RNA, allowing precise mapping of protein-RNA interactions.

What assays are recommended for measuring the pseudouridylation activity of reconstituted complexes?

For quantitative assessment of pseudouridylation activity in reconstituted H/ACA RNP complexes containing NOP10, the following protocol is recommended:

• Prepare substrate RNA labeled with 32P at specific positions

• Incubate labeled substrate with reconstituted RNP complexes at 30°C

• At defined time points, stop the reaction using phenol extraction

• Digest the modified RNA with P1 endonuclease to release mononucleotides

• Separate the mononucleotides using thin layer chromatography (TLC)

• Quantify pseudouridine formation by phosphorimaging

This methodology allows determination of both reaction kinetics and final conversion levels. Reaction conditions typically include:

Fitting the time course data to appropriate kinetic models allows extraction of parameters such as starting turnover rates (vstart) and half-maximal pseudouridylation times (t1/2) .

How can CRISPR-Cas9 be implemented for NOP10 manipulation in D. hansenii?

A CRISPR-Cas9 toolbox has recently been developed for D. hansenii, enabling precise genetic engineering of this organism . For NOP10 manipulation, the following approach is recommended:

• Design sgRNAs targeting the NOP10 gene locus with high specificity

• Prepare repair templates containing desired modifications flanked by homologous regions (30-bp minimum)

• Co-transform D. hansenii with Cas9 expression construct, sgRNA, and repair template

• Select transformants using appropriate markers

• Verify modifications through sequencing and functional assays

For complex modifications or multiple gene targets, in vivo DNA assembly can be employed. This technique allows co-transformation of up to three different DNA fragments with 30-bp homologous overlapping overhangs, which fuse in the correct order within D. hansenii cells .

What expression systems yield optimal production of recombinant D. hansenii NOP10?

For heterologous expression of D. hansenii NOP10, several systems have been evaluated. Based on available data, the following recommendations can be made:

• For expression in D. hansenii itself, the TEF1 promoter from Arxula adeninivorans combined with the CYC1 terminator provides strong expression levels

• For E. coli expression, fusion tags like His6 or GST facilitate purification while potentially enhancing solubility

• For expression in other yeasts, inducible promoters (e.g., GAL1 in S. cerevisiae) offer controlled expression

Key considerations for expression optimization include:

Post-expression purification typically involves affinity chromatography followed by size exclusion to obtain highly pure protein for reconstitution experiments.

How does NOP10 contribute to the structural stability of H/ACA RNP complexes?

NOP10 plays a critical role in the structural organization of H/ACA RNP complexes through multiple protein-protein and protein-RNA interactions. The protein forms part of the core structural backbone along with Cbf5 and Nop10, creating a platform that facilitates proper RNA conformation and catalytic activity.

In assembly experiments, the absence of NOP10 prevents the formation of stable, catalytically active complexes. This is evidenced by the significant reduction in activity observed when NOP10 is omitted from reconstitution experiments, as well as the failure of Nhp2 to bind properly to the complex in NOP10's absence .

The structural importance of NOP10 is particularly evident in the assembly of complexes on hairpin RNAs. For the 5' hairpin (H5), the combination of Cbf5-Nop10-Nhp2 results in a near-quantitative shift into a conformation that resembles the catalytically active state. This conformational change appears to involve a widening of the pseudouridylation pocket, transitioning from a "closed" state (EFRET > 0.56) to an "open" state (EFRET = 0.40) .

What conformational changes occur in H/ACA RNP complex assembly involving NOP10?

The assembly of H/ACA RNP complexes involves a series of conformational changes in both the RNA and protein components. Using smFRET analysis, these changes have been characterized in detail:

• For H5 RNA (5' hairpin):

  • In the absence of proteins or with only Nhp2 present, the RNA predominantly adopts a "closed" conformation (EFRET > 0.56)

  • Addition of the Nop10-Cbf5-Gar1 (NCG) trimer increases the population of the "open" conformation (EFRET = 0.40) to 28%

  • When all four proteins (WNCG) are present, 85% of the molecules adopt the "open" conformation

• For H3 RNA (3' hairpin):

  • The conformational behavior differs from H5, with three distinguishable FRET states observed (EFRET = 0.40, 0.62, and 0.79)

  • The refolding into a high FRET state is observable with NCG even in the absence of Nhp2

• Protein conformational changes:

  • Labeled NOP10 shows distinct FRET patterns when incorporated into the complex, indicating specific positioning within the assembled RNP

  • Different labeling positions on NOP10 (e.g., K37 vs. K48) yield similar FRET patterns, suggesting consistent protein conformation

These conformational changes appear to be prerequisites for catalytic activity, as they likely position the substrate RNA correctly within the active site of the complex.

How might the dual roles of NOP10 in RNA modification and telomere maintenance be interconnected?

The involvement of NOP10 in both pseudouridylation reactions and telomere maintenance suggests potential functional interconnections that warrant further investigation:

• RNA component modification: NOP10-containing H/ACA RNPs might modify the RNA component of telomerase, influencing its activity or stability.

• Regulatory feedback: Telomere length might influence the expression or activity of NOP10, creating a regulatory circuit.

• Protein moonlighting: NOP10 might have independent functions in telomere maintenance separate from its role in H/ACA RNPs.

• Adaptation specificity: The relationship between NOP10 and telomere maintenance might be particularly important in extremophiles like D. hansenii, where genome stability under stress conditions is crucial.

In vitro experiments have supported a mechanism in which NOP10 overexpression facilitates telomerase-dependent telomere length maintenance , but the molecular details of this relationship in D. hansenii specifically remain to be elucidated.

What methodological approaches could reveal the impact of NOP10 mutations on complex assembly and function?

To investigate the functional consequences of NOP10 mutations, a multi-faceted approach combining structural, biochemical, and cellular analyses is recommended:

• Structural analysis:

  • Introduce specific mutations in recombinant NOP10

  • Analyze complex formation using gel filtration chromatography

  • Employ smFRET to detect alterations in complex conformation

  • Use structural modeling to predict the impact of mutations

• Biochemical characterization:

  • Assess the binding affinity of mutant NOP10 to other complex components

  • Measure pseudouridylation activity of reconstituted complexes containing mutant NOP10

  • Determine the thermal and chemical stability of complexes with mutant proteins

• Cellular studies:

  • Introduce mutations into D. hansenii using CRISPR-Cas9

  • Analyze growth under varying conditions (temperature, salt concentration)

  • Examine telomere length in mutant strains

  • Assess global RNA pseudouridylation levels