Recombinant Danio rerio DNA-directed RNA polymerase III subunit RPC3 (polr3c), partial

What is the function of DNA-directed RNA polymerase III subunit RPC3 (polr3c) in Danio rerio?

POLR3C (RPC3) functions as part of a critical heterotrimer with POLR3F and POLR3G/POLR3GL that is important for transcription initiation and termination in the RNA polymerase III complex . Unlike core subunits that form the catalytic center, POLR3C belongs to peripheral subcomplexes that regulate Pol III activity. In zebrafish, as in other vertebrates, this subunit likely plays a crucial role in promoter recognition and polymerase recruitment to target genes.

Pol III is responsible for transcribing several types of small non-coding RNAs essential for cellular function, including:

How does POLR3C interact with other Pol III subunits in zebrafish?

POLR3C forms a functional heterotrimer with POLR3F and POLR3G that is crucial for proper Pol III function . This subcomplex operates distinctly from the 10-subunit core, which contains POLR3A/RPC1 and POLR3B/RPC2 that form the active site of the enzyme . The interaction between these subcomplexes is essential for proper transcription initiation and termination at Pol III promoters.

Research on RNA polymerase III in zebrafish has demonstrated that functional interactions between Pol III subunits have been highly conserved throughout eukaryotic evolution . For example, studies on the zebrafish slim jim mutant revealed that a mutation in polr3b disrupts its interaction with polr3k (the zebrafish ortholog of yeast Rpc11), affecting Pol III function and digestive organ development .

What approaches can be used to express recombinant zebrafish POLR3C for structural and functional studies?

For successful recombinant expression of zebrafish POLR3C, researchers should consider the following methodological approaches:

• Vector selection and optimization:

  • Use expression vectors with strong promoters (CMV, T7)

  • Include appropriate tags (His, FLAG, GST) for purification and detection

  • Consider codon optimization for the expression system

• Expression systems:

  • Bacterial systems: Limited by lack of post-translational modifications

  • Insect cell systems: Better for folding of complex eukaryotic proteins

  • Mammalian cell systems: Optimal for maintaining native protein interactions

• Co-expression strategies:

  • Co-express with interaction partners POLR3F and POLR3G for stability

  • Consider expressing with chaperone proteins to improve folding

• Purification protocol:

  • Use affinity chromatography based on fusion tags

  • Apply additional purification steps (ion exchange, size exclusion)

  • Verify purity and activity through biochemical assays

When designing constructs, consider the lessons from the slim jim zebrafish mutant, where a 41-amino acid deletion in Polr3b disrupted interaction with Polr3k, demonstrating the importance of preserving key interaction domains .

How can CRISPR-Cas9 genome editing be used to study POLR3C function in zebrafish?

CRISPR-Cas9 technology offers powerful approaches for investigating POLR3C function in vivo:

Based on findings from the zebrafish slim jim mutant, researchers should pay particular attention to digestive organ development when characterizing polr3c mutants, as digestive system defects have been observed with mutations in another Pol III subunit, polr3b .

What methods can be used to analyze POLR3C expression patterns during zebrafish development?

To characterize the spatiotemporal expression pattern of polr3c in zebrafish:

• Transcriptional analysis:

  • RT-qPCR to quantify expression levels at different developmental stages

  • RNA-seq for comprehensive transcriptomic profiling

  • Single-cell RNA-seq to identify cell-type specific expression patterns

• Protein detection:

  • Western blotting to track expression levels during development

  • Immunohistochemistry to visualize tissue localization

  • Generation of transgenic lines expressing tagged POLR3C

• Reporter gene approaches:

  • Create transgenic lines with the polr3c promoter driving fluorescent reporter expression

  • Use BAC transgenesis for larger regulatory regions

When designing experiments, consider that RNA polymerase III subunits may show tissue-specific functions, as suggested by the tissue-specific developmental disorders resulting from Pol III disruptions .

What developmental processes might be affected by POLR3C dysfunction in zebrafish?

Based on studies of other Pol III subunits, POLR3C dysfunction might affect several developmental processes:

These effects likely stem from the essential role of Pol III in transcribing tRNAs and 5S rRNA, which are crucial for ribosome function and protein synthesis . Tissues with high rates of protein synthesis or proliferation would be particularly vulnerable to Pol III dysfunction.

How does polr3c contribute to Pol III promoter recognition and transcription initiation?

RNA polymerase III transcribes from three distinct promoter types with varying structures and requirements for transcription initiation factors :

• Type 1 promoters (exclusive to 5S rRNA genes):

  • Utilize transcription factors TFIIIA, TFIIIB, and TFIIIC

  • POLR3C likely contributes to Pol III recruitment through interactions with these factors

• Type 2 promoters (tRNAs and some small ncRNAs):

  • Utilize TFIIIB and TFIIIC

  • POLR3C-containing heterotrimer likely facilitates recognition of these promoters

• Type 3 promoters (other ncRNAs):

  • Unique in using upstream promoter elements

  • Use a variant form of TFIIIB containing BRF2 instead of BRF1

  • Use SNAPc instead of TFIIIC

  • POLR3C may mediate specialized interactions at these promoters

The POLR3C-POLR3F-POLR3G heterotrimer likely plays a critical role in the assembly of pre-initiation complexes at all three promoter types, though with potentially different mechanisms of action at each .

How is POLR3C activity regulated during zebrafish development?

While specific information about POLR3C regulation is limited in the search results, Pol III activity is regulated through multiple mechanisms that likely affect POLR3C function:

• Transcriptional repressors:

  • MAF1 represses Pol III transcription under stress conditions

  • p53 blocks formation of basal transcription machinery complex and halts Pol III recruitment

  • RB1 blocks formation of transcription machinery by binding to BRF1 in TFIIIB

• Post-translational modifications:

  • Phosphorylation, acetylation, and sumoylation likely regulate POLR3C activity

  • These modifications may alter protein-protein interactions within the Pol III complex

• Developmental timing:

  • Expression levels likely vary throughout development

  • Interaction partners may be differentially expressed during development

Understanding how these regulatory mechanisms specifically affect POLR3C would provide insights into the precise control of Pol III function during zebrafish development.

Can overexpression of POLR3C rescue phenotypes caused by deficiencies in other Pol III subunits?

Interestingly, research on the zebrafish slim jim mutant demonstrated that phenotypic rescue through overexpression of interacting subunits is possible. Specifically, overexpression of cDNA encoding the zebrafish rpc11 ortholog, polr3k, rescued the exocrine defects in slim jim mutants, indicating that the phenotype resulted from deficiency of the Rpc11 interaction .

This suggests several experimental approaches:

• Reciprocal rescue experiments:

  • Test whether POLR3C overexpression can rescue defects in other Pol III subunit mutants

  • Determine if other subunits can compensate for POLR3C deficiency

• Structure-function analysis:

  • Create truncated or domain-mutated versions of POLR3C

  • Test which domains are necessary for functional complementation

• Cross-species rescue:

  • Test whether human POLR3C can rescue zebrafish polr3c mutants

  • Assess conservation of function across evolutionary distance

Such experiments could reveal functional redundancies and interactions within the Pol III complex that might be exploited therapeutically in Pol III-related disorders.

What human disorders are associated with POLR3C dysfunction?

While the search results don't specifically mention POLR3C-related human disorders, disruptions in Pol III function broadly result in tissue-specific developmental disorders :

Given POLR3C's role in the Pol III complex, mutations affecting this subunit could potentially contribute to similar disorders, though with potentially distinct clinical presentations based on its specific function within the complex.

How can zebrafish models of POLR3C dysfunction contribute to understanding human disease mechanisms?

Zebrafish models offer several advantages for studying POLR3C-related disease mechanisms:

• Vertebrate model with conserved Pol III structure:

  • Functional interactions between Pol III subunits are conserved in evolution

  • Allows study of complex vertebrate-specific aspects of Pol III biology

• Rapid development and optical transparency:

  • Enables real-time visualization of developmental processes

  • Facilitates high-throughput screening of genetic and chemical modifiers

• Tissue-specific phenotypes:

  • Allows investigation of how Pol III dysfunction affects different tissues

  • Provides insights into tissue-specific manifestations of human diseases

• Genetic tractability:

  • CRISPR-Cas9 enables precise manipulation of polr3c

  • Facilitates creation of disease-relevant mutations

The zebrafish slim jim mutant has already demonstrated the utility of zebrafish for studying Pol III function, revealing that disruption of polr3b affects digestive organ development through impaired interaction with polr3k . Similar approaches could be applied to study POLR3C function and related human disorders.