Recombinant Zinnia elegans Unknown protein 5

What is C. elegans PRMT-5 and how is it structurally characterized?

C. elegans PRMT-5 is a type II protein arginine methyltransferase encoded by the gene prmt-5 (C34E10.5) located on linkage group III. The protein consists of 734 amino acids with the highest sequence similarity to human PRMT5 (34% sequence identity and 48% similarity). The strongest sequence conservation occurs between residues 105-730 of C. elegans PRMT-5 and residues 58-633 of human PRMT5. The protein also shares significant homology with yeast Skb1 and Drosophila Dart1 .

What is the primary function of PRMT-5 in C. elegans?

PRMT-5 functions as a negative regulator of DNA damage-induced apoptosis in C. elegans. Unlike some other PRMTs that might affect developmental cell deaths, PRMT-5 specifically modulates apoptotic responses following DNA damage. This regulation occurs through a CEP-1/p53-dependent pathway by controlling the expression of the cell death initiator EGL-1. Inactivation of prmt-5 leads to excessive germ cell apoptosis following ionizing radiation exposure, demonstrating its crucial role in maintaining appropriate apoptotic responses to genotoxic stress .

How does PRMT-5 differ from other PRMTs in C. elegans?

While the C. elegans genome encodes six putative protein arginine methyltransferases (PRMT-1 through PRMT-6), PRMT-5 stands out for its specific role in DNA damage-induced apoptosis. Unlike other PRMTs, whose inactivation does not significantly affect developmental or damage-induced cell death profiles, PRMT-5 knockdown or mutation leads to a pronounced increase in germ cell apoptosis following γ-irradiation. This functional specificity suggests that PRMT-5 has evolved distinct regulatory roles compared to other family members in the worm .

What molecular mechanisms underlie PRMT-5's regulation of apoptosis?

PRMT-5 regulates apoptosis through a complex involving CEP-1 (the C. elegans p53 homolog) and CBP-1 (the ortholog of human p300/CBP). Biochemical analyses reveal that PRMT-5 forms a physical complex with both CEP-1 and CBP-1. While PRMT-5 interacts with CEP-1, it does not directly methylate it. Instead, PRMT-5 methylates CBP-1, which functions as a cofactor for CEP-1. This methylation likely modulates CEP-1's transcriptional activity, particularly its ability to upregulate the pro-apoptotic gene egl-1 following DNA damage. The suppression of excessive apoptosis in prmt-5 mutants upon cbp-1 knockdown supports this regulatory pathway .

How can researchers experimentally assess PRMT-5's impact on apoptosis?

Researchers should employ multiple complementary approaches to thoroughly assess PRMT-5's impact on apoptosis:

• Quantification of germ cell corpses through DIC microscopy following DNA damage (e.g., γ-irradiation)

• Acridine orange staining to visualize engulfed apoptotic cells

• Genetic epistasis analysis using mutations in core apoptosis pathway genes (e.g., ced-3, ced-4)

• Quantitative RT-PCR to measure expression of apoptosis regulators like egl-1

• Cell death time-course analysis to distinguish developmental versus damage-induced apoptosis

Controls should include both wild-type and prmt-5 mutant worms with and without DNA damage treatment. For genetic interaction studies, double mutants with genes in the apoptosis pathway should be generated and analyzed .

What experimental approaches can reveal the relationship between PRMT-5 and checkpoint signaling?

To investigate PRMT-5's relationship with DNA damage checkpoint signaling, researchers should:

• Generate double mutants of prmt-5 with checkpoint genes (e.g., hus-1, mrt-2, clk-2)

• Quantify DNA damage-induced apoptosis in these genetic backgrounds

• Analyze checkpoint activation through immunostaining for phosphorylated CHK-1

• Assess cell cycle arrest responses using BrdU incorporation or phospho-histone H3 staining

• Perform epistasis analysis to determine the hierarchical relationship between PRMT-5 and checkpoint components

The search results indicate that mutations in checkpoint genes significantly inhibit excessive germ cell death in prmt-5 mutants, suggesting that checkpoint signaling pathways are crucial for PRMT-5-mediated apoptosis regulation .

What techniques are most effective for studying PRMT-5 protein interactions?

For investigating PRMT-5 protein interactions, researchers should employ multiple complementary techniques:

• Co-immunoprecipitation (Co-IP) - Using tagged versions of PRMT-5 and potential interactors expressed in mammalian cells to detect complex formation

• GST pull-down assays - With recombinant GST-PRMT-5 and in vitro translated potential binding partners

• Yeast two-hybrid screening - To identify novel interaction partners

• Domain mapping - Using truncated protein variants to identify specific interaction domains

• Immunofluorescence co-localization - To validate interactions in cellular contexts

The search results demonstrate successful application of Co-IP and GST pull-down approaches to identify interactions between PRMT-5, CEP-1, and CBP-1. When Flag-CBP-1, Myc-PRMT-5, and Myc-CEP-1 were co-expressed in HEK293 cells, immunoprecipitation confirmed they form a complex, providing strong evidence for their functional association .

How can researchers assess PRMT-5 methyltransferase activity?

To evaluate PRMT-5 methyltransferase activity, researchers should:

• Perform in vitro methylation assays using:

  • Recombinant PRMT-5 protein

  • Potential substrate proteins (full-length or domains)

  • Radiolabeled S-adenosyl-L-methionine (³H-SAM) as methyl donor

  • SDS-PAGE resolution followed by autoradiography

• For in vivo methylation assessment:

  • Generate antibodies specific to symmetrically dimethylated arginine motifs

  • Perform immunoblotting or immunostaining to detect methylation patterns

  • Create point mutations at predicted methylation sites and assess functional consequences

The search results describe successful application of these approaches, revealing that PRMT-5 can methylate histone H4 but not H3 in vitro. Additionally, researchers identified CBP-1 as a methylation substrate, with the methylation site mapped to arginine 234 in the CBP-1(1-320) fragment .

What controls are essential when studying PRMT-5 enzymatic activity?

When investigating PRMT-5 enzymatic activity, researchers should include several critical controls:

• Enzyme-only and substrate-only reactions to rule out contamination or auto-methylation

• Catalytically inactive PRMT-5 mutants (e.g., mutations in the methyltransferase domain)

• Competitive inhibition with excess non-labeled SAM

• Substrate mutations at predicted methylation sites (e.g., arginine to alanine)

• Positive control substrates with known methylation (e.g., histone H4)

• Negative control substrates that should not be methylated

The study in the search results employed many of these controls, including testing various substrate fragments and creating point mutations. For instance, the R234A mutation in CBP-1(1-320) abrogated methylation by PRMT-5, confirming the specificity of the reaction and identifying the exact methylation site .

What genetic tools are available for studying PRMT-5 function in C. elegans?

Researchers have access to several genetic tools for studying PRMT-5 function:

• The prmt-5(gk357) deletion allele - Removes part of exon 1 and the entire exons 2 and 3, resulting in a likely strong loss-of-function mutation verified by antibody testing

• RNA interference (RNAi) - For tissue-specific or conditional knockdown of prmt-5

• Reporter gene constructs - To monitor expression patterns and transcriptional activity

• Double mutant analysis - For genetic interaction studies with apoptosis regulators and checkpoint genes

• Transgenic rescue lines - For structure-function analysis and tissue-specific rescue

The prmt-5(gk357) mutant exhibits normal development except for a slightly reduced growth rate but shows pronounced hypersensitivity to DNA damage-induced apoptosis, making it a valuable tool for mechanistic studies .

How can researchers differentiate between direct and indirect effects of PRMT-5 on apoptosis?

To distinguish direct from indirect effects of PRMT-5 on apoptosis, researchers should:

• Perform detailed time-course analyses of apoptotic events following DNA damage

• Monitor immediate transcriptional changes of apoptotic regulators (e.g., egl-1)

• Use ChIP assays to determine if PRMT-5 and its partners directly associate with regulatory regions of apoptotic genes

• Create domain-specific mutations in PRMT-5 to separate different functions

• Identify and characterize all components in the PRMT-5/CEP-1/CBP-1 complex

• Perform tissue-specific rescue experiments to determine where PRMT-5 function is required

The research shows that loss of prmt-5 leads to specific over-upregulation of the cell death initiator EGL-1 following DNA damage, suggesting a direct regulatory relationship. The physical interaction between PRMT-5, CEP-1, and CBP-1 further supports a direct regulatory mechanism rather than indirect effects .

What approaches can resolve contradictory findings about PRMT-5 substrates across species?

To resolve contradictions regarding PRMT-5 substrates across different species, researchers should:

• Perform rigorous comparative biochemical analyses using recombinant proteins from multiple species

• Conduct phylogenetic analysis of substrate conservation and methylation motifs

• Employ mass spectrometry to identify and compare methylation sites

• Create chimeric proteins to map species-specific substrate recognition domains

• Use in vivo cross-species complementation experiments

The search results highlight potential species differences, as C. elegans PRMT-5 methylates histone H4 but not H3 in vitro, while mammalian PRMT5 methylates both H4R3 and H3R8. Additionally, unlike in mammals, C. elegans PRMT-5 does not appear to significantly affect global H4R3 symmetric dimethylation in vivo, suggesting evolutionary divergence in substrate specificity or regulatory mechanisms .

How should researchers design experiments to study PRMT-5 in cancer-related processes?

When investigating PRMT-5's potential role in cancer-related processes, researchers should:

• Compare PRMT-5 expression and activity between normal and cancer cell models

• Analyze PRMT-5's impact on cell cycle regulation, apoptotic resistance, and DNA damage responses

• Identify transcriptional targets that might link PRMT-5 to tumorigenesis (e.g., tumor suppressors)

• Investigate PRMT-5's interactions with known cancer-associated proteins

• Employ genetic models with tissue-specific PRMT-5 alterations to study cancer development in vivo

The search results note that aberrant expression of PRMT5 is associated with various cancer types including lymphoma, leukemia, gastric carcinoma, and testicular tumors. In lymphoma, PRMT5 overexpression correlates with increased H4R3 and H3R8 symmetric dimethylation, potentially suppressing the tumor suppressor gene ST7. These findings suggest that PRMT-5's role in cancer involves transcriptional regulation of key genes .

What methodological approaches can reveal the full spectrum of PRMT-5 substrates?

To comprehensively identify PRMT-5 substrates, researchers should employ multiple complementary approaches:

• Proteome-wide analyses using:

  • Antibodies specific to symmetrically dimethylated arginine motifs

  • Stable isotope labeling with amino acids in cell culture (SILAC)

  • Mass spectrometry to identify methylated proteins

• Candidate-based approaches testing:

  • Proteins that physically interact with PRMT-5

  • Proteins containing conserved methylation motifs

  • Functional homologs of known PRMT5 substrates from other species

• Validation strategies including:

  • In vitro methylation assays with recombinant proteins

  • Site-directed mutagenesis of predicted methylation sites

  • Functional tests to determine the significance of specific methylation events

The search results demonstrate partial application of these approaches, identifying CBP-1 as a novel PRMT-5 substrate through interaction studies and in vitro methylation assays .

How can researchers best investigate the evolutionary conservation of PRMT-5 function?

To study evolutionary conservation of PRMT-5 function, researchers should:

• Perform comparative sequence analysis across species to identify conserved domains and motifs

• Test cross-species complementation (e.g., express human PRMT5 in C. elegans prmt-5 mutants)

• Compare biochemical activities of PRMT-5 from different species using identical substrates

• Identify and compare PRMT-5 interaction partners across species

• Analyze conservation of PRMT-5-regulated pathways (e.g., apoptosis, transcriptional regulation)

The search results illustrate evolutionary conservation in PRMT-5 function, as both C. elegans PRMT-5 and human PRMT5 negatively regulate p53-dependent apoptosis involving CBP/p300 proteins. This suggests fundamental conservation of the regulatory mechanism despite some differences in substrate specificity .

What are the optimal conditions for recombinant PRMT-5 production and purification?

For efficient recombinant PRMT-5 production and purification, researchers should:

• Express PRMT-5 as a fusion protein (e.g., GST or His-tagged) to facilitate purification

• Consider using eukaryotic expression systems (insect or mammalian cells) for proper folding

• Optimize expression conditions (temperature, induction duration, media composition)

• Include protease inhibitors during purification to prevent degradation

• Perform activity assays to confirm the enzymatic function of purified protein

• Store purified protein with glycerol at -80°C in small aliquots to maintain activity

The search results describe successful production of recombinant PRMT-5 that retained methyltransferase activity against histone H4 in vitro, demonstrating the feasibility of producing functional recombinant protein for biochemical studies .

What considerations are important when interpreting prmt-5 mutant phenotypes?

When analyzing prmt-5 mutant phenotypes, researchers should consider:

• The nature of the mutation (null, hypomorph, conditional)

• Potential redundancy with other PRMTs or compensatory mechanisms

• Tissue-specific effects versus systemic impacts

• Developmental timing of phenotype manifestation

• Environmental conditions that may enhance or suppress phenotypes

• The specific readouts being measured (e.g., apoptosis, gene expression)

The search results indicate that prmt-5(gk357) is likely a strong loss-of-function allele based on antibody testing, yet it shows normal development except for slightly reduced growth rate. This suggests either functional redundancy or a non-essential role during development. The mutant's specific hypersensitivity to DNA damage-induced apoptosis highlights the importance of examining phenotypes under both normal and stress conditions .

How can researchers effectively measure changes in gene expression regulated by PRMT-5?

To accurately measure PRMT-5-regulated gene expression changes, researchers should:

• Perform RNA-seq or microarray analysis to identify global transcriptional changes

• Validate key targets using quantitative RT-PCR with appropriate reference genes

• Use reporter constructs to monitor transcriptional activity of specific promoters

• Conduct ChIP assays to identify direct binding sites of PRMT-5 or its partners

• Compare expression profiles across different tissues and developmental stages

• Analyze expression changes under both normal and stress conditions

The research demonstrates effective application of quantitative RT-PCR to measure egl-1 expression in wild-type and prmt-5 mutant worms following DNA damage. This approach revealed that loss of prmt-5 leads to excessive upregulation of egl-1, directly linking PRMT-5 to transcriptional regulation of this pro-apoptotic gene .

How might PRMT-5 function integrate with other post-translational modifications?

To investigate how PRMT-5-mediated arginine methylation interacts with other post-translational modifications, researchers should:

• Perform proteome-wide analyses to identify proteins subject to both PRMT-5 methylation and other modifications

• Test whether pre-existing modifications affect PRMT-5 substrate recognition

• Determine if PRMT-5 methylation influences subsequent modifications

• Examine potential crosstalk between histone arginine methylation and other histone modifications

• Create modification-specific antibodies to monitor combinatorial modification patterns

The search results do not directly address this question, but the identification of CBP-1 as a PRMT-5 substrate raises interesting possibilities, as CBP-1 possesses acetyltransferase activity. This suggests potential crosstalk between arginine methylation and acetylation pathways in regulating transcription and apoptosis .

What techniques can reveal PRMT-5's potential role in non-transcriptional processes?

To explore PRMT-5's functions beyond transcriptional regulation, researchers should:

• Perform subcellular fractionation to identify PRMT-5 localization in different cellular compartments

• Use proximity labeling approaches (BioID, APEX) to identify context-specific interaction partners

• Examine potential roles in RNA processing, translation, or protein stability

• Investigate PRMT-5's impact on cellular structures and organelles using microscopy

• Analyze post-transcriptional processes in prmt-5 mutants compared to wild-type

While the search results focus primarily on PRMT-5's role in transcriptional regulation of apoptosis, the identification of non-histone substrates like CBP-1 suggests PRMT-5 likely has broader functions. The study notes that a genome-wide RNAi screen previously found that inactivation of prmt-5 causes increased levels of spontaneous mutation, indicating potential roles in genome stability beyond transcriptional regulation .

How can new technologies advance our understanding of PRMT-5 function in vivo?

Emerging technologies that could enhance PRMT-5 research include:

• CRISPR/Cas9 gene editing for:

  • Creating precise mutations in PRMT-5 catalytic domains

  • Generating fluorescently tagged endogenous PRMT-5

  • Developing conditional knockout models

• Single-cell technologies for:

  • Analyzing cell-type-specific responses to PRMT-5 inactivation

  • Identifying rare cell populations affected by PRMT-5 loss

  • Mapping temporal dynamics of PRMT-5-regulated processes

• Advanced imaging approaches for:

  • Tracking PRMT-5 localization and dynamics in live cells

  • Visualizing PRMT-5-containing complexes at endogenous levels

  • Monitoring methylation events in real-time