Recombinant Chicken CCR4-NOT transcription complex subunit 10 (CNOT10), partial

What is the structural composition of chicken CNOT10 and how does it compare to mammalian orthologs?

Chicken (Gallus gallus) CNOT10 is composed entirely of tetratricopeptide (TPR) repeats, forming a soluble protein of approximately 707 amino acids (from residue M24 to Q707) . The protein's structure is highly conserved across species, showing remarkable similarity to human CNOT10, with both proteins adopting similar TPR-based solenoid structures. Cryo-EM studies revealed that chicken CNOT10 forms part of a ternary complex with NOT1 and NOT11, with structural features conserved from avian to mammalian systems .

The TPR repeats of CNOT10 create a specialized structural scaffold that facilitates protein-protein interactions within the CCR4-NOT complex. These repeats are arranged to form binding surfaces that accommodate other subunits, particularly NOT11, which binds to the C-terminal region of CNOT10. This structural arrangement enables the assembly of the functional NOT10:11 module of the complex .

What expression systems are most effective for producing recombinant chicken CNOT10?

For efficient production of recombinant chicken CNOT10, researchers have successfully employed bacterial expression systems using specialized plasmid constructs. Specifically, cDNA fragments encoding chicken (Gg) NOT10 (residues M24–Q707) can be cloned into bicistronic plasmids based on the pnEA backbone . This approach allows for the expression of untagged NOT10, which maintains its native folding properties.

When co-expression with other complex components is desired, such as with NOT11, a dual-plasmid system can be advantageous. In published protocols, researchers have used a plasmid encoding chicken NOT1 N-terminus (residues M1–N682) without a solubility tag, alongside another plasmid encoding both NOT10 and NOT11 . This coordinated expression strategy facilitates the formation of the ternary complex directly during protein production, potentially enhancing stability and solubility of the recombinant proteins.

How can I verify the integrity and functionality of purified recombinant chicken CNOT10?

Verification of recombinant chicken CNOT10 integrity requires a multi-faceted approach combining biochemical and functional assays. After purification, size-exclusion chromatography can confirm the proper oligomeric state of the protein, as properly folded CNOT10 should elute at a volume consistent with its monomeric molecular weight unless it's co-purified with binding partners .

Functional verification can be achieved through interaction studies with known binding partners, particularly NOT11 and NOT1. Pulldown assays using purified recombinant proteins provide a reliable method to confirm that chicken CNOT10 maintains its ability to form complexes . The interaction between CNOT10 and NOT11 is particularly crucial, as this heterodimer formation is a prerequisite for subsequent binding to NOT1. Therefore, demonstrating this hierarchical assembly pattern through pulldown experiments serves as an excellent functional verification for recombinant chicken CNOT10 .

What is the hierarchical assembly mechanism of the NOT10:11 module, and how do molecular interactions stabilize this structure?

The assembly of the NOT10:11 module follows a distinct hierarchical pattern where NOT11 first binds to NOT10, which then organizes the heterodimer for subsequent binding to NOT1. Biochemical dissection studies across multiple species, including chicken, human, and Drosophila, have revealed that this assembly mechanism is evolutionarily conserved . The C-terminal portion of NOT10 is primarily responsible for the interaction with NOT11, while the NOT11-NOT10 heterodimer as a unit establishes contact with NOT1 .

A critical molecular determinant in this assembly is a short proline-rich motif in NOT11 that functions as a molecular stabilizer for the entire module. This motif, which has been termed the NOT10-binding motif (NOT10-BM), interacts with the C-terminal TPR repeats of NOT10 and helps organize the locally flexible TPR solenoid of NOT10 to form the NOT1-binding interface . Mutational studies demonstrate that replacing key proline residues in this motif with alanines abolishes the interaction with NOT10, highlighting the essential role of this motif in module assembly . This mechanism exemplifies how a linear peptide motif can act as a molecular determinant of an entire module assembly, representing an elegant solution for complex protein architecture stabilization.

What methodological approaches are most effective for studying the interactions between chicken CNOT10 and other CCR4-NOT subunits?

For investigating interactions between chicken CNOT10 and other CCR4-NOT subunits, a combination of structural, biochemical, and biophysical techniques has proven most informative. Cryo-electron microscopy (cryo-EM) represents a powerful approach, with researchers achieving sub-3 Å resolution structures of the chicken NOT1:NOT10:NOT11 ternary complex . This technique reveals the spatial arrangement of subunits and identifies key interaction interfaces that can be further investigated through targeted mutagenesis.

Biochemical interaction studies using recombinantly expressed protein constructs provide complementary information about binding requirements and domain contributions. For instance, pulldown assays with systematically truncated protein constructs have been instrumental in mapping interaction domains and identifying the minimal regions necessary for stable complex formation . These experiments revealed that while the C-terminal portion of NOT10 interacts with NOT11, the NOT10:NOT11 heterodimer is required for efficient binding to NOT1, as isolated NOT10 does not bind NOT1 effectively .

Additionally, mutational analysis targeting conserved residues at interaction interfaces provides critical insights into the molecular determinants of complex assembly. By replacing key residues with alanines and assessing the impact on complex formation, researchers can identify essential amino acids that stabilize inter-subunit interactions . This approach has been particularly valuable for characterizing the role of the proline-rich motif in NOT11 that mediates binding to NOT10.

How does the structural flexibility of the NOT10:11 module contribute to its function within the CCR4-NOT complex?

The NOT10:11 module exhibits notable structural flexibility that appears integral to its functional role within the CCR4-NOT complex. Cryo-EM studies of both chicken and human complexes reveal that while certain regions of the module show well-defined density, others display considerable flexibility . For instance, the HEAT C domain of NOT1 is disordered in the chicken complex, indicating flexibility and suggesting a peripheral role in supporting module stability .

Similarly, the NOT11 MIF4G domain is absent in the density maps of both chicken and human structures, consistent with a high degree of flexibility for this domain . This structural plasticity may enable the module to accommodate interaction with diverse binding partners or adopt different conformations during various functional states of the CCR4-NOT complex.

The connection between structural flexibility and function is particularly evident in how the rigid proline-rich motif of NOT11 organizes the locally flexible TPR solenoid of NOT10 to form the NOT1-binding interface . This molecular arrangement suggests that controlled flexibility is crucial for the module's assembly and integration within the larger CCR4-NOT complex. The evolutionary conservation of this flexible architecture across species from insects to vertebrates underscores its functional significance .

What are the optimal expression and purification protocols for obtaining stable recombinant chicken CNOT10 suitable for structural studies?

Obtaining stable recombinant chicken CNOT10 for structural studies requires careful optimization of expression and purification protocols. For expression, bicistronic plasmids based on the pnEA backbone have been successfully used to express chicken NOT10 (residues M24–Q707) . When co-expressing with NOT11, it is recommended to design constructs that express untagged NOT10 alongside NOT11 with a C-terminal, TEV-cleavable 6xHis tag . This approach facilitates complex formation during expression and enables affinity purification through the tagged partner.

The expression system should be optimized for temperature, induction conditions, and duration to maximize protein yield while maintaining proper folding. Typically, lower temperatures (16-18°C) after induction favor proper folding of complex proteins like CNOT10 . For purification, a multi-step approach is recommended, beginning with affinity chromatography utilizing the His-tag on NOT11. Following tag cleavage with TEV protease, size exclusion chromatography serves to isolate properly assembled complexes and remove aggregates or improperly folded species .

For structural studies requiring high purity and homogeneity, additional purification steps might be necessary. Ion exchange chromatography can help separate species with different surface charge distributions, while limited proteolysis can be employed to remove flexible regions that might hinder crystallization or introduce heterogeneity in cryo-EM samples . The final buffer composition should be optimized for stability during storage and for compatibility with the intended structural technique, whether it's cryo-EM, crystallography, or biophysical characterization methods.

How can I design experiments to investigate the functional consequences of disrupting the NOT10:11 module in cellular contexts?

To implement these mutations in cellular contexts, CRISPR-Cas9 gene editing can be employed to introduce specific mutations in the endogenous NOT11 gene. Alternatively, a knockdown-rescue approach can be utilized, where endogenous NOT11 is depleted using RNA interference, followed by expression of either wild-type or mutant NOT11 constructs. This approach allows for direct comparison between cells expressing functional versus disrupted NOT10:11 modules .

Functional readouts should assess various aspects of CCR4-NOT function, including mRNA deadenylation rates, global gene expression patterns through RNA-seq, and transcript-specific decay through pulse-chase experiments. Since the NOT10:11 module enhances CCR4-NOT deadenylation activity in vitro , assays measuring poly(A) tail length distributions and deadenylation kinetics would be particularly informative. Additionally, ribosome profiling could reveal effects on translation, as the NOT10:11 module may provide a link between CCR4-NOT and ribosomes .

What approaches can be used to identify potential protein interaction partners specific to chicken CNOT10 compared to mammalian orthologs?

Identifying protein interaction partners specific to chicken CNOT10 requires comparative interactomics approaches that distinguish between conserved and species-specific interactions. Affinity purification coupled with mass spectrometry (AP-MS) represents a powerful technique for this purpose. Researchers can express tagged versions of chicken CNOT10 in both avian cell lines (such as DF-1 chicken fibroblasts) and mammalian cells (such as human HEK293), followed by pulldown and mass spectrometric identification of co-purifying proteins .

Comparative analysis of the resulting interaction networks can reveal proteins that preferentially associate with chicken CNOT10 over its mammalian counterparts. To control for non-specific interactions, quantitative proteomics approaches such as SILAC (Stable Isotope Labeling with Amino acids in Cell culture) or TMT (Tandem Mass Tag) labeling can be employed . These techniques enable statistical evaluation of interaction specificity and help distinguish true interactors from background proteins.

Complementary to mass spectrometry-based approaches, proximity labeling methods such as BioID or TurboID offer the advantage of capturing transient or context-dependent interactions. By fusing chicken CNOT10 to a biotin ligase enzyme, proteins in close proximity become biotinylated and can subsequently be purified and identified . Cross-species comparative proximity labeling would highlight interaction partners unique to the avian ortholog. Validation of identified partners should employ orthogonal techniques such as co-immunoprecipitation, yeast two-hybrid assays, or in vitro binding studies with recombinant proteins .

How does chicken CNOT10 contribute to the deadenylation activity of the CCR4-NOT complex, and how can this be measured experimentally?

To experimentally measure CNOT10's contribution to deadenylation, researchers can employ in vitro deadenylation assays using reconstituted CCR4-NOT complexes with or without the NOT10:11 module. These assays typically utilize synthetic RNA substrates with defined poly(A) tails that are radiolabeled or fluorescently tagged . The rate of poly(A) tail shortening can be monitored over time through gel electrophoresis, with the difference in deadenylation rates between complete complexes and those lacking CNOT10 quantifying its contribution to activity.

For cellular contexts, pulse-chase experiments with 4-thiouridine (4sU) labeling can track the decay rates of endogenous mRNAs in cells expressing wild-type versus mutant CNOT10, or in CNOT10 knockdown conditions . Additionally, poly(A) tail length profiling using techniques such as TAIL-seq or PAL-seq can provide genome-wide measurements of poly(A) tail lengths, revealing the impact of CNOT10 perturbation on global deadenylation patterns . These approaches would elucidate whether chicken CNOT10's contribution to deadenylation is uniform across all mRNAs or preferentially affects specific transcripts.

What are the evolutionary implications of the conserved NOT10:11 module structure among vertebrates, and how can comparative studies inform our understanding of CCR4-NOT function?

The remarkable conservation of the NOT10:11 module structure from insects to vertebrates, including chicken and humans, suggests fundamental importance for CCR4-NOT function that has been maintained throughout evolution . Comparative studies across species reveal that while the molecular architecture of the module is preserved, subtle species-specific adaptations may tune its function to particular cellular environments or regulatory networks.

Structural comparisons between chicken and human NOT10:11 modules show high conservation of the interaction interfaces, particularly the binding mechanism between NOT11 and NOT10, which is mediated by a conserved NOT10-binding motif (NOT10-BM) . This conserved interaction mode extends beyond vertebrates to invertebrates like Drosophila, and potentially to plants and fungi, highlighting its ancient evolutionary origin . This conservation contrasts with some other CCR4-NOT subunits like NOT4, which is stably associated with the complex in yeast but only loosely associated in humans .

Comparative functional studies can explore whether species-specific differences in the NOT10:11 module correlate with divergent regulatory mechanisms or target specificities. For instance, researchers might investigate whether chicken CNOT10 recognizes the same RNA-binding proteins or regulatory factors as its mammalian orthologs . By identifying both conserved and divergent aspects of NOT10:11 function across species, such comparative approaches would provide insights into the fundamental versus specialized roles of this module in post-transcriptional regulation throughout evolution.

How can structural information about chicken CNOT10 inform the development of tools to study post-transcriptional regulation mechanisms?

The detailed structural information available for chicken CNOT10 within the NOT10:11 module provides a valuable foundation for developing molecular tools to study post-transcriptional regulation mechanisms . By leveraging the high-resolution cryo-EM structures showing the precise interatomic contacts between CNOT10 and its binding partners, researchers can design rational structure-based tools that specifically target or modulate these interactions.

One approach involves developing dominant-negative mutants of CNOT10 that can assemble with endogenous partners but disrupt specific functions. For example, mutations could be introduced that maintain NOT11 binding but prevent subsequent interaction with NOT1, effectively sequestering NOT11 away from the functional complex . Such tools would allow for selective perturbation of NOT10:11 module functions while leaving other CCR4-NOT activities intact, enabling more precise dissection of post-transcriptional regulatory mechanisms.

The structural identification of the proline-rich motif in NOT11 as a critical determinant for module assembly suggests another potential tool development strategy . Short peptides mimicking this motif could be designed to competitively inhibit NOT10:11 interaction, providing a chemical approach to acutely disrupt the module. Additionally, the development of conformation-specific antibodies that recognize assembled versus disassembled states of the NOT10:11 module would facilitate tracking of complex dynamics during various cellular processes .

For studying the spatial and temporal dynamics of CCR4-NOT function, the structural information could guide the design of fluorescent protein fusions or split fluorescent protein complementation systems that minimize interference with complex assembly . By strategically positioning tags based on structural data, researchers can develop imaging tools that report on complex formation, localization, and activity in living cells, advancing our understanding of how post-transcriptional regulation is coordinated in time and space.

What strategies can address the solubility challenges often encountered when expressing recombinant chicken CNOT10?

Recombinant chicken CNOT10 expression often faces solubility challenges due to its complex structure composed entirely of tetratricopeptide (TPR) repeats . To address these issues, co-expression with interaction partners represents one of the most effective strategies. The NOT10:11 module forms through hierarchical assembly, with NOT11 binding to CNOT10, which then organizes it for binding to NOT1 . Co-expressing chicken CNOT10 with at least NOT11, if not the entire ternary complex including NOT1, can significantly improve solubility by stabilizing the native conformation during protein folding.

Optimization of expression conditions can also substantially improve solubility. Lowering the induction temperature to 16-18°C, reducing IPTG concentration, and extending expression time can favor proper folding over rapid production . Additionally, the choice of expression vector and fusion tags can dramatically impact solubility. While some studies have successfully expressed untagged chicken CNOT10 , others have benefited from solubility-enhancing tags such as MBP (maltose-binding protein) or SUMO. When using tags, incorporating a cleavage site allows for their removal after solubilization.

Another approach involves construct optimization based on structural knowledge. Full-length chicken CNOT10 (residues M24–Q707) may be less soluble than carefully designed truncations that preserve essential functional domains while removing regions prone to aggregation . Bioinformatic analysis identifying structured domains and potentially disordered regions can guide this rational construct design. Finally, screening different buffer conditions during purification, including various pH values, salt concentrations, and stabilizing additives like glycerol or mild detergents, can significantly improve the recovery of soluble protein after expression.

How can researchers address the challenge of structural heterogeneity in chicken CNOT10 samples prepared for cryo-EM analysis?

Structural heterogeneity presents a significant challenge for cryo-EM analysis of chicken CNOT10, particularly when studying it as part of larger assemblies like the NOT10:11 module. Cryo-EM studies of chicken NOT1:NOT10:NOT11 complexes have revealed considerable flexibility in certain regions, with the HEAT C domain of NOT1 appearing disordered and the NOT11 MIF4G domain absent from density maps . This heterogeneity can reduce the achievable resolution and complicate structural interpretation.

To address this challenge, researchers can employ biochemical approaches to reduce sample heterogeneity. Limited proteolysis can remove flexible regions that contribute to conformational variability while preserving the core structured domains . By systematically testing different proteases and conditions, followed by mass spectrometry analysis of the resulting fragments, researchers can identify stable core domains that may yield more homogeneous samples. Additionally, cross-linking strategies using mild chemical cross-linkers can "lock" the complex in more uniform conformations, reducing structural heterogeneity.

From a data processing perspective, advanced computational approaches can help manage heterogeneity during cryo-EM analysis. Techniques such as 3D classification allow for sorting particles into distinct conformational states, enabling reconstruction of multiple structures from a single dataset . Moreover, focused refinement approaches can improve resolution for well-ordered regions by computationally masking flexible portions during image processing. For highly dynamic complexes, methods such as manifold embedding or multi-body refinement can model continuous conformational changes, providing insights into the range of structural states adopted by the NOT10:11 module.

What control experiments are essential when investigating chicken CNOT10's role in deadenylation using in vitro and cellular assays?

When investigating chicken CNOT10's role in deadenylation, rigorous control experiments are essential to establish specificity and rule out artifacts. For in vitro deadenylation assays, a crucial control is the comparison between catalytically active and inactive CCR4-NOT complexes. This can be achieved by introducing point mutations in the catalytic residues of the CCR4 and CAF1 deadenylase subunits, which should abolish activity regardless of CNOT10's presence . This control verifies that observed deadenylation truly depends on the catalytic subunits and is not due to contaminating nucleases.

When testing the enhancement of deadenylation by CNOT10, comparisons should include not only complexes with and without CNOT10 but also complexes with mutant CNOT10 variants that maintain complex incorporation but disrupt specific functions . For instance, mutations that prevent NOT11 binding while maintaining incorporation into the larger complex would help distinguish between CNOT10's structural role versus potential regulatory functions. Additionally, titration experiments with increasing concentrations of CNOT10 or the NOT10:11 module can establish dose-dependence of any observed effects, strengthening the evidence for a direct role .

For cellular assays, controls should account for potential indirect effects of CNOT10 perturbation. When using knockdown or knockout approaches, rescue experiments with wild-type CNOT10 are essential to confirm that observed phenotypes are directly attributable to CNOT10 loss rather than off-target effects . Monitoring the integrity of the remaining CCR4-NOT complex after CNOT10 depletion is also crucial, as loss of CNOT10 might destabilize other subunits, complicating interpretation. Finally, when assessing deadenylation of specific transcripts, controls should include mRNAs known to be regulated by different deadenylase complexes, helping to establish the specificity of CNOT10's contribution to CCR4-NOT-mediated deadenylation versus general effects on mRNA decay pathways .