Recombinant Cricetulus griseus Nuclear nucleic acid-binding protein C1D (C1D)

What is the nuclear nucleic acid-binding protein C1D in Cricetulus griseus?

C1D in Cricetulus griseus is a small nuclear matrix protein (approximately 16 kDa) that demonstrates high-affinity binding to both DNA and RNA . It belongs to the C1D family of proteins, which includes yeast homologues Rrp47 (in Saccharomyces cerevisiae) and Cti1 (in Schizosaccharomyces pombe) . The protein contains a Sas10/C1D domain that likely functions as a docking site for simultaneous interactions with RNA and DNA . C1D is ubiquitously expressed in mammalian tissues and localizes primarily to the nucleus, with significant accumulation in the nucleolus where it participates in RNA processing activities .

What are the primary cellular functions of C1D protein?

C1D serves as a multifunctional protein positioned at the intersection of several critical cellular processes:

• RNA processing: C1D interacts with the nuclear exosome complex, particularly binding to PM/Scl-100 (the human homolog of yeast Rrp6), to facilitate 5.8S rRNA maturation and other RNA processing events .

• DNA damage repair: C1D participates in the repair of DNA double-strand breaks (DSBs) through both non-homologous end joining (NHEJ) and homologous recombination (HR) pathways .

• Chromatin architecture regulation: Similar to its yeast homologue Cti1, C1D likely interacts with condensin complexes to influence higher-order chromatin structure .

• Apoptosis induction: When DNA damage is beyond repair, C1D can trigger p53-dependent apoptosis, functioning as a cellular safeguard against genomic instability .

How conserved is the C1D protein sequence and function across species?

The C1D protein family demonstrates notable evolutionary conservation, particularly in the Sas10/C1D domain. While sequence conservation exists between mammalian C1D and its yeast homologues (Rrp47 and Cti1), their molecular weights differ (16 kDa for human C1D versus 21 kDa for yeast Rrp47) . Despite these differences, functional conservation is evident in their shared roles in RNA processing and DNA repair pathways .

All members of this protein family can bind both DNA and RNA, interact with exosome components, and contribute to maintaining genomic stability . The Cricetulus griseus C1D is expected to share significant homology with human C1D, given the conservation of this protein across mammalian species and the use of Chinese hamster ovary (CHO) cells as experimental models for mammalian cell processes .

What expression systems are optimal for producing recombinant Cricetulus griseus C1D protein?

For the recombinant expression of Cricetulus griseus C1D, several expression systems can be employed with specific advantages:

• E. coli expression system: This provides high yields but may require optimization for proper folding of mammalian proteins. For C1D expression, BL21(DE3) strains with pET-based vectors incorporating a His-tag for purification are recommended. Expression should be induced at lower temperatures (16-20°C) to enhance proper folding .

• Mammalian expression systems: CHO cells themselves represent an excellent homologous expression system for Cricetulus griseus C1D. Using vectors with CMV promoters and incorporating epitope tags (FLAG, HA) facilitates detection and purification while maintaining native post-translational modifications .

• Baculovirus expression system: This system offers advantages for proper folding and post-translational modifications while providing higher yields than mammalian systems. For C1D, which functions in complex protein-protein and protein-nucleic acid interactions, this system may preserve functional activity better than bacterial expression .

The choice of expression system should be guided by the intended experimental applications, with bacterial systems favored for structural studies requiring large quantities, and mammalian systems preferred for functional studies where proper folding and modifications are critical.

What purification strategies yield functional recombinant C1D protein?

To obtain pure, functional recombinant C1D protein from Cricetulus griseus, a multi-step purification strategy is recommended:

• Affinity chromatography: Using His-tag, GST-tag, or other fusion tags allows for initial capture of the recombinant protein. For C1D, which binds nucleic acids, it's essential to include DNase/RNase treatments and high-salt washes (>500 mM NaCl) to remove bound nucleic acids that might co-purify .

• Ion exchange chromatography: As a second step, this helps remove contaminants with different charge characteristics. C1D typically has a basic isoelectric point, making cation exchange chromatography appropriate .

• Size exclusion chromatography: This final polishing step separates aggregates and provides a homogeneous preparation of monomeric C1D protein.

The purified protein should be assessed for functionality through:

• Nucleic acid binding assays (EMSA)

• Interaction studies with known binding partners (e.g., PM/Scl-100)

• Activity assays measuring contribution to relevant RNA processing reactions

Buffer composition is critical, with typical buffers containing 20-50 mM Tris or HEPES (pH 7.5-8.0), 150-300 mM NaCl, 1-5 mM DTT, and 10% glycerol to maintain stability .

How can researchers validate the functional activity of recombinant C1D protein?

Validating the functional activity of recombinant Cricetulus griseus C1D requires multiple complementary approaches:

• RNA processing functionality:

  • In vitro RNA processing assays using specific exosome substrates

  • Co-immunoprecipitation with exosome components (especially PM/Scl-100)

  • 5.8S rRNA maturation assays in cells depleted of endogenous C1D and reconstituted with recombinant protein

• DNA repair functionality:

  • In vitro DNA binding assays (EMSA)

  • DNA-PK phosphorylation assays (C1D is an efficient substrate for DNA-PK)

  • Complementation assays in C1D-depleted cells exposed to DNA damaging agents

• Structural integrity validation:

  • Circular dichroism spectroscopy to confirm proper folding

  • Size exclusion chromatography to verify monomeric state

  • Limited proteolysis to assess domain organization integrity

A functional validation table comparing wild-type and mutant recombinant C1D can provide valuable insights:

What is the specific role of C1D in the repair of DNA double-strand breaks?

C1D plays critical roles in both major pathways of DNA double-strand break repair:

• Non-homologous end joining (NHEJ):

  • C1D interacts with DNA-dependent protein kinase (DNA-PK), a key enzyme in NHEJ .

  • It appears to be involved in the 3'-end processing step, which is crucial for proper joining of DNA ends .

  • C1D may suppress error-prone DSB repair pathways, as studies of its yeast homologue Rrp47 showed approximately 50% inappropriate repair of 3'-overhanging ends in mutants .

  • The interaction with DNA-PK occurs through DNA-PK's leucine zipper (LZ) region, although some studies suggest this binding may be more complex .

• Homologous recombination (HR):

  • Rrp47 mutants (yeast homologue of C1D) showed a two-fold reduction in HR repair efficiency .

  • While not as essential as proteins like Rad52, C1D appears to contribute to the fidelity and efficiency of HR pathways .

  • C1D likely helps maintain genome integrity by reducing mutation introduction during repair .

• Regulatory role:

  • C1D may function at the intersection of RNA processing and DNA repair, potentially coordinating these processes at sites of transcription-associated DNA damage .

  • When DNA damage is beyond repair, C1D can trigger p53-dependent apoptosis, serving as a quality control mechanism .

The dual involvement of C1D in both NHEJ and HR suggests it may function in pathway choice or in ensuring repair fidelity across multiple mechanisms.

How does C1D coordinate RNA processing with DNA repair activities?

C1D occupies a strategic position at the interface between RNA processing and DNA repair pathways, making it an ideal coordinator of these processes:

• Co-localization at damage sites:

  • C1D, through its ability to bind both RNA and DNA, may recognize transcription-associated DNA damage .

  • The nucleolar localization of C1D positions it to respond to nucleolar stress, which often triggers the redistribution of repair factors .

• Temporal coordination:

  • Evidence suggests that proper activation of DNA damage response (DDR) pathways requires post-transcriptional mechanisms that regulate mRNA processing and metabolism .

  • C1D may facilitate the temporary suspension of RNA processing at sites of DNA damage to allow repair machinery access .

• Mechanistic links:

  • Pre-mRNA splicing factors prevent exon skipping and safeguard against DNA damage, with DNA repair proteins providing regulatory feedback to control splicing processes .

  • C1D, with its dual functionality, may ensure that transcription-coupled repair occurs efficiently at active genes .

• R-loop management:

  • R-loops (RNA-DNA hybrids) form during transcription and can lead to genomic instability if not properly managed.

  • C1D may help resolve R-loops through its interaction with the exosome, preventing R-loop-mediated DNA damage .

This coordination is particularly important at highly transcribed loci, where the collision between transcription and replication machinery can lead to genomic instability if not properly regulated.

What experimental approaches best reveal C1D's function in DNA repair?

To comprehensively investigate C1D's functions in DNA repair, multiple complementary approaches should be employed:

• Cell-based assays:

  • CRISPR-Cas9 knockout or knockdown of C1D in Chinese hamster cells followed by various DNA damage treatments (ionizing radiation, radiomimetic drugs, enzymatic induction of DSBs)

  • Reporter assays for specific repair pathways (e.g., DR-GFP for HR, EJ5-GFP for NHEJ)

  • Immunofluorescence to track C1D localization to DNA damage sites

  • Cell survival and proliferation assays following DNA damage in C1D-deficient cells

• Biochemical approaches:

  • In vitro reconstitution of NHEJ and HR with purified components including recombinant C1D

  • DNA binding assays to characterize C1D's affinity for different DNA structures (blunt ends, overhangs, nicks)

  • Immunoprecipitation following DNA damage to identify damage-specific interaction partners

  • Phosphorylation assays to study C1D as a substrate for DNA-PK and potentially other DNA damage response kinases

• Advanced microscopy:

  • Live-cell imaging of fluorescently tagged C1D during DNA damage response

  • Super-resolution microscopy to visualize C1D localization at individual damage sites

  • FRAP (Fluorescence Recovery After Photobleaching) to measure C1D dynamics at damage sites

• Genomic approaches:

  • ChIP-seq to map C1D binding sites genome-wide before and after DNA damage

  • RNA-seq in C1D-deficient cells to identify transcriptional changes affecting DNA repair gene expression

  • END-seq or BLESS to map DSBs genome-wide in C1D-deficient cells

How does C1D contribute to RNA exosome function in Chinese hamster cells?

Based on studies of C1D homologues, the role of Cricetulus griseus C1D in RNA exosome function likely involves:

• Exosome recruitment and stabilization:

  • C1D binds directly to PM/Scl-100 (the mammalian homolog of yeast Rrp6), an exosome-associated 3'-5' exoribonuclease .

  • This interaction appears necessary for C1D to enter the nucleus and accumulate in the nucleolus .

  • C1D likely stabilizes PM/Scl-100, as seen with its yeast homolog Rrp47, which maintains Rrp6 protein levels .

• RNA substrate recognition and processing:

  • The C1D protein, along with hMPP6 and hMtr4, forms a complex with PM/Scl-100 that is required for 5.8S rRNA maturation .

  • C1D likely helps the exosome recognize specific RNA substrates for processing .

  • The complex provides redundant exonuclease activities for the 3' end maturation of specific RNAs, similar to the function of Rrp47 in box C/D snoRNA maturation in yeast .

• Nucleolar RNA processing:

  • C1D accumulates in the nucleolus, which houses machinery for rRNA synthesis and ribosome assembly .

  • It likely participates in quality control mechanisms for nucleolar RNAs .

  • The protein may facilitate the processing of cryptic unstable transcripts (CUTs) and other non-coding RNAs, as suggested by the function of its yeast homolog .

Unlike in yeast, where depletion of either Rrp47 or Rrp6 results in similar RNA processing defects, the relationship between C1D and PM/Scl-100 in mammalian cells may be more complex and involve additional regulatory layers specific to higher eukaryotes.

What methodologies are effective for studying C1D-mediated RNA processing?

To investigate C1D-mediated RNA processing in Chinese hamster cells, researchers should employ these methodologies:

• RNA processing assays:

  • Northern blot analysis to detect specific RNA species (e.g., 5.8S rRNA precursors) in C1D-depleted cells

  • Pulse-chase labeling with 4-thiouridine to track newly synthesized RNA processing

  • In vitro reconstitution of RNA processing reactions using purified components

  • RNA immunoprecipitation (RIP) to identify RNAs directly bound by C1D

• Protein interaction studies:

  • Co-immunoprecipitation to identify C1D interaction partners in Chinese hamster cells

  • Proximity labeling (BioID or APEX) to capture transient interactions in the RNA processing complexes

  • Yeast two-hybrid or mammalian two-hybrid assays to map interaction domains

  • Size-exclusion chromatography combined with western blotting to define native complex composition

• Localization studies:

  • Immunofluorescence to track C1D localization, particularly in the nucleolus

  • Cell fractionation to biochemically separate nuclear, nucleolar, and cytoplasmic components

  • Electron microscopy with immunogold labeling for high-resolution localization

• Functional genomics:

  • CRISPR-Cas9 knockout of C1D followed by RNA-seq to identify globally affected transcripts

  • CLIP-seq (Crosslinking and Immunoprecipitation followed by sequencing) to map RNA binding sites of C1D

  • Structure probing methods (e.g., SHAPE-seq) to examine RNA structural changes in the presence/absence of C1D

A systematic approach combining these methodologies provides comprehensive insights into how C1D contributes to RNA processing in Chinese hamster cells, potentially revealing species-specific aspects of its function.

How can researchers generate C1D mutants to dissect its multiple functions?

To dissect the multiple functions of Cricetulus griseus C1D, researchers should generate targeted mutations based on structural and functional domains:

• DNA binding mutants:

  • Create point mutations in positively charged residues likely involved in DNA binding

  • Design truncation mutants to remove DNA-binding regions while preserving other functional domains

  • Test these mutants in DNA binding assays (EMSA) and DNA repair complementation studies

• RNA binding mutants:

  • Identify residues in the Sas10/C1D domain potentially involved in RNA interactions

  • Generate mutants with altered RNA binding specificity but maintained protein-protein interactions

  • Validate using RNA binding assays and RNA processing complementation experiments

• PM/Scl-100 interaction mutants:

  • Create mutations at the interface between C1D and PM/Scl-100

  • Design truncation mutants that specifically disrupt this interaction

  • Confirm through co-immunoprecipitation and exosome activity assays

• DNA-PK interaction mutants:

  • Mutate residues involved in DNA-PK binding, particularly those interacting with the leucine zipper region

  • Generate phosphorylation-site mutants (serine/threonine to alanine) to prevent DNA-PK-mediated phosphorylation

  • Test in DNA-PK binding assays and DNA repair functional studies

A comprehensive mutational analysis can be summarized in the following table:

What are the most challenging aspects of C1D research and how can they be addressed?

Research on Cricetulus griseus C1D presents several significant challenges with corresponding solutions:

• Distinguishing multiple functions:
  Challenge: C1D's involvement in both RNA processing and DNA repair makes it difficult to separate these functions experimentally.
  Solution: Use cell-type specific conditional knockouts combined with function-specific rescue experiments. Employ synchronized cell populations to separate cell cycle-dependent functions. Create separation-of-function mutants as described in section 5.1.

• Protein stability issues:
  Challenge: Like its yeast homolog Rrp47, C1D stability may depend on interaction partners such as PM/Scl-100 .
  Solution: Co-express C1D with stabilizing partners during recombinant production. Use degradation-resistant fusion tags when studying C1D in isolation. Design experiments that account for interdependent protein stability.

• Phenotype attribution:
  Challenge: Phenotypes observed after C1D depletion could result from either direct C1D function or indirect effects through its interaction partners.
  Solution: Use rapid protein degradation systems (e.g., auxin-inducible degron) to distinguish immediate from long-term effects. Perform targeted rescue experiments with specific interaction-deficient mutants.

• Redundancy with other factors:
  Challenge: Functional redundancy, as seen between Rrp47 and Mpp6 in yeast , may mask C1D-specific phenotypes.
  Solution: Generate double or triple knockouts of redundant factors. Use chemical genetic approaches to simultaneously inhibit redundant pathways. Perform studies under stress conditions that may eliminate functional redundancy.

• Species-specific differences:
  Challenge: Extrapolating from yeast or human studies to Chinese hamster C1D may miss species-specific functions.
  Solution: Perform comparative functional studies with C1D from multiple species in the same experimental system. Use domain-swapping experiments to identify species-specific functional elements.

How can multi-omics approaches enhance understanding of C1D functions?

Multi-omics strategies provide powerful approaches to comprehensively analyze C1D functions in Cricetulus griseus cells:

• Integrative genomics:

  • ChIP-seq to map C1D binding sites genome-wide

  • CLIP-seq or PAR-CLIP to identify RNA binding sites

  • Integration of these datasets to identify loci where C1D interacts with both DNA and RNA

  • Correlation with gene expression data to identify functional consequences of binding

• Transcriptomics and RNA processing:

  • RNA-seq in C1D-depleted cells to identify affected transcripts

  • 3'-seq to specifically examine effects on RNA 3' end processing

  • Nascent RNA sequencing (e.g., PRO-seq) to distinguish transcriptional from post-transcriptional effects

  • Small RNA-seq to examine effects on non-coding RNA processing

• Proteomics approaches:

  • Proximity-dependent biotinylation (BioID/TurboID) coupled with mass spectrometry to identify the C1D interactome

  • Quantitative proteomics following C1D depletion to identify protein abundance changes

  • Phosphoproteomics to examine C1D-dependent signaling pathways

  • Cross-linking mass spectrometry to map interaction interfaces between C1D and its partners

• Structural biology integration:

  • Cryo-EM structures of C1D-containing complexes

  • Hydrogen-deuterium exchange mass spectrometry to map dynamic interactions

  • Integrative structural modeling combining multiple data types

An integrated multi-omics workflow for C1D research might include:

• Generate CRISPR knockout or knockdown CHO cell lines targeting C1D

• Perform RNA-seq, ChIP-seq, and proteomics under normal and stress conditions

• Identify key affected pathways and direct targets

• Validate findings with targeted biochemical and cell biology experiments

• Develop mathematical models predicting C1D's role in coordinating RNA processing and DNA repair

This approach would yield a systems-level understanding of how C1D coordinates its multiple functions to maintain genomic stability in Chinese hamster cells.