Recombinant Bovine Cooperator of PRMT5 (COPR5)

What is Cooperator of PRMT5 (COPR5) and what are its primary functions?

COPR5 is a nuclear protein that functions as an important adaptor for Protein Arginine Methyltransferase 5 (PRMT5). It serves as a critical bridge between PRMT5 and chromatin, facilitating the recruitment of PRMT5 to specific genomic regions. COPR5 tightly binds to PRMT5 both in vitro and in living cells, but notably does not interact with other members of the PRMT family .

The primary functions of COPR5 include:

• Modulating the substrate specificity of nuclear PRMT5-containing complexes toward histones

• Recruiting PRMT5 to chromatin at specific target genes

• Binding directly to the amino terminus of histone H4

• Contributing to gene expression regulation, particularly for genes like cyclin E1 (CCNE1)

What is the molecular structure and characterization of bovine COPR5?

Bovine COPR5, like its human counterpart, is a relatively small protein rich in acidic residues. Human COPR5 spans 184 amino acids and shows neither a canonical protein domain nor significant similarity with other proteins . The bovine variant shares high sequence homology with other mammalian COPR5 proteins.

Key structural features include:

• A conserved PRMT5 Binding Motif (PBM) with the consensus sequence GQF[D/E]DA[D/E]

• The last 44 C-terminal residues are essential for both PRMT5 binding and histone interaction

• Nuclear localization signals that direct the protein to the nucleus

What are the recommended methods for recombinant expression and purification of bovine COPR5?

To successfully express and purify recombinant bovine COPR5, researchers should consider the following methodology:

• Expression System Selection: Bacterial expression systems (E. coli) work effectively for COPR5 production, typically using BL21(DE3) strains with pGEX vectors for GST-tagged protein or pET vectors for His-tagged protein.

• Purification Strategy:

  • For GST-tagged COPR5: Use glutathione-Sepharose affinity chromatography

  • For His-tagged COPR5: Use Ni-NTA affinity chromatography

  • Implement size exclusion chromatography as a secondary purification step to ensure high purity

• Quality Control: Confirm protein identity and purity through:

  • SDS-PAGE analysis (COPR5 migrates at approximately 20-25 kDa depending on tags)

  • Western blotting using specific antibodies

  • Mass spectrometry verification

How can researchers effectively assess COPR5-PRMT5 interactions in vitro?

Multiple complementary approaches can be used to characterize COPR5-PRMT5 interactions:

• Pull-down Assays: GST-COPR5 efficiently pulls down both in vitro-translated PRMT5 and endogenous PRMT5 from nuclear extracts. In contrast, it does not pull down other PRMTs like PRMT1 or PRMT4, confirming interaction specificity .

• Co-immunoprecipitation: Both ectopically expressed and endogenous PRMT5 can be co-immunoprecipitated with Flag-COPR5, confirming their association in nuclear extracts .

• Glycerol Gradient Size Fractionation: Probing fractions with PRMT5 and COPR5 antibodies demonstrates that endogenous proteins distribute in overlapping high-molecular-weight fractions, suggesting they are components of the same nuclear complex .

• Yeast Two-hybrid Assays: This approach confirms direct protein-protein interactions and can determine if COPR5 interacts with enzymatically inactive forms of PRMT5 .

How does COPR5 influence PRMT5-mediated histone methylation patterns?

COPR5 significantly alters the substrate specificity of PRMT5 toward histones in the following ways:

• Preferential Methylation: PRMT5 bound to COPR5 preferentially methylates histone H4 (R3) compared to histone H3 (R8), indicating that COPR5 modulates the substrate specificity of nuclear PRMT5-containing complexes .

• Histone Binding Capacity: COPR5 functions as a histone-binding protein with affinity comparable to the archetypal histone-binding protein HP1. It can efficiently pull down histone H3 and H4 dimers from purified histones .

• Specificity for H4: Overlay experiments with N-terminal H3 and H4 peptides confirm that COPR5 directly interacts with H4 but not H3. Notably, neither R3 dimethylation nor K-acetylation affects this histone H4-COPR5 interaction .

• Bridging Function: COPR5 serves as an adaptor protein that bridges PRMT5 to chromatin, similar to how HP1 connects nucleosomal histones to the lysine methyltransferase SUV39H1 .

What is the impact of COPR5 depletion or overexpression on gene regulation?

COPR5 levels significantly affect gene expression patterns through several mechanisms:

• CCNE1 Expression Regulation:

  • COPR5 depletion via siRNA leads to a less than twofold increase (1.8×) in CCNE1 mRNA levels

  • Infection with a retroviral vector encoding COPR5 results in significant reduction in CCNE1 mRNA levels

  • These effects are not observed for all PRMT5 target genes (e.g., NM23 mRNA levels remain unchanged), suggesting COPR5 is involved in only some nuclear functions of PRMT5

• Promoter Activity Effects:

  • shCOPR5-mediated depletion potentiates E2F/DP1-mediated transactivation of the CCNE1 promoter

  • COPR5 overexpression markedly decreases E2F-stimulated transcription of the CCNE1 reporter

  • These effects are not observed with CCNE1 promoter constructs mutated at the CERC-binding site (CERM) or with E2F site-driven synthetic promoters that do not respond to PRMT5

• Chromatin Recruitment: COPR5 depletion strongly reduces PRMT5 recruitment to chromatin at target genes, leading to decreased H4R3me2s marks in those regions .

How can researchers effectively analyze COPR5 recruitment to chromatin in vivo?

Chromatin immunoprecipitation (ChIP) is the gold standard technique for analyzing COPR5 chromatin association:

• ChIP Protocol Optimization:

  • Crosslinking: Use 1% formaldehyde for 10 minutes at room temperature

  • Sonication: Optimize conditions to generate DNA fragments of 200-500 bp

  • Immunoprecipitation: Use validated antibodies against COPR5, PRMT5, and histone modifications (e.g., H4R3me2s)

  • Controls: Include IgG and other chromatin-associated proteins (e.g., HP1) as controls

• Target Region Selection: Focus on specific nucleosome regions, such as the nucleosome 12 (nu12) region of the CCNE1 promoter, which contains CERM and the transcription start site .

• Sequential ChIP: To demonstrate co-occupancy of COPR5 and PRMT5 on the same DNA fragments, perform sequential ChIP (re-ChIP) experiments using antibodies against both proteins.

• Data Analysis and Quantification: Normalize ChIP data to input DNA and compare enrichment between target regions and control regions to determine specific binding .

What approaches can be used to study the evolutionarily conserved PRMT5 Binding Motif (PBM) in COPR5?

The PRMT5 Binding Motif (PBM) with the consensus sequence GQF[D/E]DA[D/E] is crucial for COPR5 function. Researchers can study this motif using:

• Mutagenesis Studies:

  • Generate point mutations in key residues within the PBM

  • Create deletion constructs that remove the PBM

  • Assess the impact on PRMT5 binding, histone interaction, and functional outcomes

• Peptide Competition Assays:

  • Synthesize peptides corresponding to the wild-type and mutant PBM sequences

  • Test whether these peptides can compete with full-length COPR5 for PRMT5 binding

  • Determine the minimal peptide sequence required for interaction

• Structural Biology Approaches:

  • X-ray crystallography or cryo-EM studies of COPR5-PRMT5 complexes

  • NMR studies of the interaction interface

  • In silico molecular modeling to predict interaction surfaces

What are the key functional domains of COPR5 and how do they contribute to its activity?

COPR5 contains several functional regions that are essential for its various activities:

The C-terminal region (last 44 amino acids) is particularly important as it serves a dual function in both PRMT5 binding and histone interaction, consistent with COPR5's role as a bridging molecule between PRMT5 and chromatin .

Beyond PRMT5, what other protein interactions are significant for COPR5 function?

While PRMT5 is the primary interaction partner for COPR5, several other proteins and complexes may associate with COPR5:

• Histone Proteins: COPR5 directly binds to histone H4 at its N-terminal region, with binding affinity comparable to HP1 .

• Transcriptional Regulators: Given COPR5's role in regulating the CCNE1 promoter, it likely interacts with transcription factors such as those in the E2F family and components of the CERC (Cyclin E Repressor Module) complex .

• Chromatin Remodeling Complexes: COPR5 may associate with chromatin remodeling complexes, similar to how PRMT5 interacts with the human SWI-SNF complex for regulation of genes such as NM23 .

• Other Epigenetic Modifiers: While not directly shown in the available data, COPR5 may functionally interact with other epigenetic modifiers that co-regulate gene expression at PRMT5 target genes.

How conserved is COPR5 across species, and what does this tell us about its evolutionary importance?

COPR5 demonstrates a specific pattern of evolutionary conservation that provides insights into its biological significance:

The restricted taxonomic distribution of COPR5 to tetrapods suggests it may have evolved to fulfill specialized roles in higher vertebrates, possibly related to complex gene regulation patterns required for tetrapod-specific developmental and physiological processes.

How do the functions of bovine COPR5 compare to human and other mammalian COPR5 proteins?

Based on available research, bovine COPR5 appears to share fundamental characteristics with its human counterpart:

• Core Functions: Both bovine and human COPR5 serve as adaptor proteins for PRMT5, facilitating its recruitment to chromatin and modulating its substrate specificity toward histones .

• Molecular Interactions: The key interactions with PRMT5 and histone H4 are preserved across mammalian species, mediated by the conserved C-terminal region containing the PBM .

• Gene Regulation: While specific target genes may vary between species, the mechanistic role of COPR5 in transcriptional regulation through PRMT5-mediated histone modification appears consistent .

• Structural Features: The relatively small size, acidic nature, and domain organization are conserved features across mammalian COPR5 proteins .

The high degree of functional conservation makes bovine COPR5 a relevant model for understanding human COPR5 biology, particularly in fundamental aspects of epigenetic regulation and chromatin dynamics.

How can COPR5 research contribute to our understanding of epigenetic dysregulation in disease states?

COPR5 research has significant implications for understanding disease mechanisms through several pathways:

• Cancer Biology: Given that PRMT5 is a therapeutic target in MTAP-null cancers, understanding the role of COPR5 in modulating PRMT5 activity may reveal new therapeutic approaches. Disruption of the PRMT5-substrate adaptor interface impairs growth of MTAP-null tumor cells, suggesting a potential target for therapeutic development .

• Cell Cycle Regulation: COPR5's involvement in regulating CCNE1 (cyclin E1) expression connects it to cell cycle control, a process frequently dysregulated in cancer and other proliferative disorders .

• Epigenetic Reprogramming: As a mediator of histone arginine methylation, COPR5 may influence broader epigenetic landscapes relevant to cellular differentiation, development, and disease progression.

• Splicing Regulation: Disruption of the PRMT5-substrate adaptor interface affects Sm spliceosome activity, leading to intron retention, which has implications for diseases associated with aberrant RNA processing .

What methodological challenges persist in studying COPR5-PRMT5 interactions in complex chromatin environments?

Several technical challenges remain in fully understanding COPR5-PRMT5 interactions within chromatin:

• Dynamic Range of Interactions: Capturing the temporal dynamics of COPR5-PRMT5-chromatin interactions during gene regulation remains technically challenging. Advanced approaches like time-resolved ChIP-seq or real-time imaging of tagged proteins may help address this limitation.

• Context-dependent Functions: COPR5 appears to be involved in some but not all nuclear functions of PRMT5, suggesting context-dependent roles that are difficult to decipher using traditional biochemical approaches alone .

• Nucleosome-specific Effects: Understanding how nucleosome positioning and existing histone modifications influence COPR5 binding requires specialized techniques like nucleosome positioning assays combined with COPR5 binding studies.

• Interplay with Other Adaptors: PRMT5 utilizes multiple adaptor proteins (CLNS1A, RIOK1, and COPR5) that share a common binding motif. Determining how these adaptors compete or cooperate for PRMT5 binding in vivo requires sophisticated proteomics and imaging approaches .

• Sub-nuclear Localization: Resolving the precise sub-nuclear localization of COPR5-PRMT5 complexes requires super-resolution microscopy techniques that can distinguish between different nuclear compartments.

What emerging technologies could advance our understanding of COPR5 function?

Several cutting-edge technologies show promise for deepening our understanding of COPR5 biology:

• CRISPR-based Epigenome Editing: Using catalytically inactive Cas9 (dCas9) fused to COPR5 or PRMT5 to target specific genomic loci and assess direct effects on chromatin modification and gene expression.

• Proximity Labeling Techniques: BioID or APEX2-based approaches to identify proximal proteins to COPR5 in living cells, potentially revealing novel interaction partners beyond PRMT5.

• Single-molecule Tracking: Visualizing the dynamics of individual COPR5 molecules in live cells to understand its mobility, residence time on chromatin, and interaction kinetics with PRMT5.

• Cryo-electron Microscopy: Resolving the structure of COPR5-PRMT5 complexes bound to nucleosomes at high resolution to understand the molecular details of these interactions.

• Multi-omics Integration: Combining ChIP-seq, RNA-seq, and proteomics data to build comprehensive models of COPR5-dependent gene regulatory networks and their functional consequences.

What are the most promising areas for future COPR5 research in bovine systems?

Several research directions appear particularly promising for advancing our understanding of bovine COPR5:

• Developmental Biology: Investigating COPR5's role in bovine embryonic development, particularly during phases requiring extensive epigenetic reprogramming.

• Comparative Genomics: Conducting comparative studies between bovine and human COPR5 to identify species-specific functional adaptations and conserved regulatory mechanisms.

• Agricultural Applications: Exploring how COPR5-mediated epigenetic regulation influences economically important traits in cattle, potentially informing breeding strategies.

• Disease Models: Developing bovine cell culture models to study how COPR5 dysfunction contributes to disease states, which may have translational relevance to human health.

• Environmental Epigenetics: Investigating how environmental factors influence COPR5-dependent epigenetic marks in bovine cells, contributing to our understanding of gene-environment interactions.