Recombinant Xenopus laevis PRKR-interacting protein 1 homolog (prkrip1)

What is PRKRIP1 and what is its primary function in Xenopus laevis?

PRKRIP1 (PRKR-interacting protein 1 homolog) in Xenopus laevis functions primarily as a component of the spliceosome and is required for pre-mRNA splicing. It has the ability to bind double-stranded RNA . The protein contains specific functional domains that enable these interactions, similar to its homologs in other vertebrates. While most studies of PRKRIP1 have been conducted in mammalian systems, the Xenopus homolog shares significant sequence conservation, suggesting conserved functionality across vertebrate species.

How does Xenopus laevis PRKRIP1 compare with its homolog in Xenopus tropicalis?

Xenopus tropicalis PRKRIP1 consists of 173 amino acids and has the sequence: MAAETGTAKPARAKKEPQPLVIPKNATDEQRLRLERLMRNPDKLAAIPERP KEWSPRSAPEFVRDVMGSSAGAGSGEFHVYRHLRRREYQRQEFLDRVSEKHNI DDDFQRKLMENKKLEEETKAKRRLKRQKLKEKKKMAKMAKKEEKKEEIEKLVELDN SPESSDKSLEDQ . Xenopus laevis, being allotetraploid compared to the diploid X. tropicalis, may have two copies of the prkrip1 gene with slight variations. Cross-species analysis shows that both are annotated with similar functions in RNA processing and binding. In X. tropicalis, the gene is also known by the synonyms c114 and krbox3 .

What are the known protein domains and structural features of Xenopus PRKRIP1?

While the complete structural characterization of Xenopus PRKRIP1 is not fully documented in the provided research, the protein is known to contain domains necessary for RNA binding and protein-protein interactions essential for its spliceosomal function . Based on mammalian research, PRKRIP1 likely contains regions that facilitate interaction with the RNA-dependent protein kinase (PKR) and regions involved in nucleic acid binding. The protein's ability to bind double-stranded RNA suggests structural elements that recognize and interact with RNA secondary structures.

What is the expression pattern of PRKRIP1 during Xenopus development?

While specific developmental expression data for PRKRIP1 is not detailed in the provided materials, Xenopus laevis developmental gene expression can be analyzed using resources like Xenbase that maintain cataloged expression data across developmental stages . The normal table of Xenopus development shows that gene expression patterns can be examined from fertilized egg through metamorphosis , and PRKRIP1 would follow a specific pattern related to its function in RNA processing. Similar proteins involved in fundamental cellular processes like splicing typically show expression throughout development but may have stage-specific regulation.

How is PRKRIP1 expression regulated in Xenopus oocytes and embryos?

While the specific regulation of PRKRIP1 in Xenopus oocytes and embryos is not explicitly covered in the provided research, studies have shown that microRNAs can regulate translation in Xenopus oocytes through AGO and FXR1 proteins . Given that PRKRIP1 is involved in RNA processing, its expression might be regulated post-transcriptionally. The deep proteomics analysis of Xenopus laevis eggs has identified correlation between mRNA and protein abundance (Pearson correlation of 0.32) , suggesting complex regulatory mechanisms for proteins like PRKRIP1.

What are the best methods for expressing recombinant Xenopus PRKRIP1?

For recombinant expression of Xenopus PRKRIP1, several systems have proven effective:

• Yeast expression system: This is economical and efficient for eukaryotic protein expression, providing proper post-translational modifications such as glycosylation, acylation, and phosphorylation to ensure native protein conformation .

• Xenopus oocyte expression system: For functional studies, Xenopus oocytes themselves provide an excellent system for heterologous protein expression. Following microinjection of cRNA into oocytes, the protein can be expressed and studied in a near-native environment .

• Cell-free protein synthesis: This approach can be used for rapid production of PRKRIP1 without cellular constraints.

For structural studies, the purification of recombinant proteins expressed in Xenopus laevis oocytes has been optimized to avoid contamination from egg yolk lipids, phospholipids, and lipoproteins .

What purification strategies are most effective for recombinant Xenopus PRKRIP1?

Effective purification of recombinant Xenopus PRKRIP1 typically involves:

• Affinity chromatography: Using multi-tagged proteins (such as His-tagged PRKRIP1) for efficient purification . The His tag allows for isolation via nickel or cobalt affinity resins.

• Lipid removal: A critical step when purifying from Xenopus oocytes is efficiently discarding lipids, phospholipids, and lipoproteins from the oocyte egg yolk, which are major contaminants in protein purifications .

• Buffer optimization: For PRKRIP1, Tris-based buffers with 50% glycerol have been used for storage of the purified protein .

The protein should be stored at -20°C for extended storage, with working aliquots kept at 4°C for up to one week. Repeated freezing and thawing should be avoided .

How can I design functional assays to study PRKRIP1 activity in Xenopus systems?

To study PRKRIP1 function in Xenopus systems, consider these methodological approaches:

• RNA binding assays: Since PRKRIP1 binds double-stranded RNA , electrophoretic mobility shift assays (EMSAs) with labeled RNA substrates can detect binding activity.

• Splicing assays: As PRKRIP1 functions in pre-mRNA splicing , in vitro splicing assays using Xenopus egg extracts with depleted or supplemented PRKRIP1 can assess its role.

• Oocyte expression system for functional characterization: Xenopus oocytes provide an excellent system for expression and functional characterization of proteins . This approach allows:

  • Microinjection of PRKRIP1 cRNA into oocytes

  • Assessment of protein function through radiotracer assays

  • Determination of functionally relevant residues and domains

• Developmental studies: Morpholino-mediated knockdown or CRISPR-Cas9 gene editing in Xenopus embryos can reveal developmental roles of PRKRIP1, following approaches similar to those used for studying other proteins in Xenopus development .

What is the role of PRKRIP1 in immune responses in Xenopus?

The potential immune function of PRKRIP1 in Xenopus is an intriguing research area that remains to be fully explored. Studies on Receptor Transporter Proteins (RTPs) in Xenopus laevis have shown that they play roles in antiviral responses . Given that PRKRIP1 (PRKR-interacting protein 1) interacts with PKR, which is a key antiviral protein in mammals that recognizes viral double-stranded RNA, PRKRIP1 might modulate immune responses in Xenopus.

RNA sequencing has revealed that many Xenopus RTPs are upregulated following immune stimulation , and similar analyses could determine if PRKRIP1 follows similar expression patterns. The ability of PRKRIP1 to bind double-stranded RNA further suggests potential roles in recognizing viral nucleic acids as part of innate immune responses.

How does PRKRIP1 interact with the Xenopus spliceosome complex?

As a component of the spliceosome, PRKRIP1 in Xenopus is required for pre-mRNA splicing . While the specific interactions within the Xenopus spliceosome are not fully characterized in the provided research, studies could employ:

• Co-immunoprecipitation coupled to mass spectrometry: This approach has been successful in identifying protein interactions in Xenopus egg extracts . Similar methodology could identify PRKRIP1's binding partners in the spliceosome.

• Chromatin immunoprecipitation: This could determine if PRKRIP1 associates with specific genomic regions or RNA species during splicing.

• Structural analysis: While not yet reported for Xenopus PRKRIP1, structural studies similar to those done for other Xenopus proteins could elucidate interaction surfaces with other spliceosomal components.

The functional importance of these interactions could be tested through mutational analysis and functional splicing assays in Xenopus oocyte or egg extract systems.

What is known about PRKRIP1's role in developmental RNA processing in Xenopus?

During Xenopus development, significant transcriptomic changes occur, particularly at the maternal-to-zygotic transition . As a component of the spliceosome , PRKRIP1 likely plays a role in processing transcripts during this crucial developmental period. Research on regenerating Xenopus embryos has identified differential expression of RNA processing factors during early development , suggesting that proteins like PRKRIP1 may have developmentally regulated functions.

To investigate this further, researchers could employ:

• RNA-seq analysis across developmental stages with PRKRIP1 knockdown or overexpression

• Analysis of alternative splicing patterns in the presence/absence of PRKRIP1

• Examination of PRKRIP1 binding partners during different developmental stages

How does PRKRIP1 function compare between Xenopus and mammalian systems?

While detailed comparative analysis of PRKRIP1 function between Xenopus and mammals is not provided in the research materials, several insights can be drawn:

• Conserved function: The basic function of PRKRIP1 in RNA binding and splicing appears to be conserved between Xenopus and mammals .

• Protein interaction networks: In mammals, PRKRIP1 interacts with PKR (Protein Kinase R), which is involved in the antiviral response. The Xenopus homolog likely maintains this interaction, though this may need experimental verification.

• Evolutionary conservation: Deep proteomics studies in Xenopus have shown that many proteins share functionality with their mammalian counterparts despite some sequence divergence .

• Expression systems: Xenopus oocytes have been used successfully to express both Xenopus and mammalian proteins for functional studies , suggesting conserved protein folding and functionality.

A detailed comparison would require experimental approaches including:

• Side-by-side functional assays of both proteins in the same system

• Cross-species rescue experiments

• Structural studies to compare protein domains and interaction surfaces

What are common challenges in producing recombinant Xenopus PRKRIP1 and how can they be overcome?

Researchers working with recombinant Xenopus PRKRIP1 may encounter several challenges:

• Protein solubility issues: PRKRIP1, as an RNA-binding protein, may have solubility challenges. Solutions include:

  • Optimizing expression conditions (temperature, induction time)

  • Using solubility-enhancing tags (MBP, SUMO)

  • Testing different buffer compositions during purification

• Contamination with egg yolk components: When purifying from Xenopus oocytes, a major challenge is contamination with lipids, phospholipids, and lipoproteins. The solution involves developing specific protocols for efficiently removing these contaminants .

• Proper folding: Ensuring proper protein folding is critical for functional studies. Using eukaryotic expression systems like yeast can help ensure proper post-translational modifications and folding .

• Storage stability: PRKRIP1 should be stored in Tris-based buffer with 50% glycerol at -20°C, avoiding repeated freeze-thaw cycles .

How can I optimize PRKRIP1 expression in the Xenopus oocyte system?

To optimize expression of recombinant PRKRIP1 in Xenopus oocytes:

• RNA quality: Ensure high-quality, capped and polyadenylated cRNA for microinjection.

• Injection protocol:

  • Inject 2-10 ng of cRNA per oocyte

  • Allow 2-4 days for protein expression

  • Maintain oocytes at 18°C in appropriate buffer

• Codon optimization: Consider using codon-optimized sequences for Xenopus expression.

• Expression verification: Use Western blotting with anti-tag antibodies to confirm expression.

• Functional verification: For PRKRIP1, RNA binding assays can verify functionality.

The expression system has been successfully used for various proteins including membrane proteins, making it suitable for PRKRIP1 expression .

What approaches are most effective for studying PRKRIP1 interactions with other proteins in Xenopus?

To study PRKRIP1 interactions with other proteins in Xenopus systems:

• Co-immunoprecipitation coupled to mass spectrometry: This approach has successfully identified protein interactions in Xenopus egg extracts . For example, Yap interacting proteins were identified in S-phase egg extracts, revealing factors functionally associated with mRNA metabolic processes .

• Reciprocal co-IP assays: These can confirm specific interactions, as demonstrated for Yap/Rif1 interactions in egg extracts .

• Heterologous expression systems: Validating interactions following expression of tagged proteins in systems like HEK293 cells can complement findings from Xenopus systems .

• Functional validation: Using targeted protein depletion approaches like Trim-Away in early embryonic development can validate functional significance of identified interactions .

• Imaging approaches: Immunostaining experiments in Xenopus tissues can confirm co-expression and co-localization of interacting proteins .

These approaches can reveal PRKRIP1's interaction network related to its roles in splicing, RNA binding, and potential immune functions.

How can CRISPR-Cas9 gene editing be used to study PRKRIP1 function in Xenopus?

CRISPR-Cas9 gene editing offers powerful approaches to study PRKRIP1 function in Xenopus:

• Knockout studies: Generating prkrip1 knockout Xenopus enables assessment of its essential functions. This approach can be particularly valuable given the allotetraploid nature of Xenopus laevis, which may have gene redundancy .

• Tagging endogenous PRKRIP1: Inserting fluorescent protein tags or epitope tags into the endogenous locus allows tracking of PRKRIP1 localization and dynamics during development.

• Domain-specific mutations: Introducing specific mutations to functional domains can assess their roles in PRKRIP1 function.

• Temporal control: Combining CRISPR with inducible systems permits temporal control of gene disruption, allowing study of PRKRIP1 function at specific developmental stages.

CRISPR experiments in Xenopus typically involve microinjection of sgRNAs and Cas9 protein or mRNA into fertilized eggs, with analysis of resulting phenotypes and molecular changes during development.

What is the potential role of PRKRIP1 in the maternal-to-zygotic transition in Xenopus?

The maternal-to-zygotic transition (MZT) is a critical period in Xenopus development involving massive changes in the transcriptome and proteome. As a protein involved in RNA processing, PRKRIP1 may play important roles during this transition:

• Processing of maternal mRNAs: PRKRIP1 might be involved in processing stored maternal mRNAs that direct early development. Studies have shown that DNA replication dynamics change during early embryonic development , which may involve differential RNA processing.

• Activation of zygotic genome: As the zygotic genome becomes active, PRKRIP1 could participate in processing newly transcribed pre-mRNAs.

• Regulation by microRNAs: MicroRNAs can activate translation in Xenopus oocytes , and PRKRIP1 could be regulated by similar mechanisms during MZT.

• Interaction with maternal factors: PRKRIP1 might interact with maternal factors stored in the egg to regulate early development before zygotic genome activation.

Research approaches could include temporal analysis of PRKRIP1 expression and binding partners across MZT stages, and functional studies using knockdown or overexpression followed by transcriptomic analysis.

How might PRKRIP1 contribute to antiviral defense mechanisms in Xenopus?

Given PRKRIP1's ability to bind double-stranded RNA and its interaction with PKR (a known antiviral protein in mammals), it potentially contributes to antiviral defense in Xenopus:

• Recognition of viral nucleic acids: PRKRIP1 might participate in detecting viral double-stranded RNA, similar to how RTPs in Xenopus contribute to antiviral responses .

• Modulation of immune signaling: Analysis of RTPs in Xenopus has shown that they can inhibit RNA virus infection . PRKRIP1 might similarly modulate antiviral signaling pathways.

• Regulation of splicing during infection: As a spliceosome component , PRKRIP1 could regulate alternative splicing of immune-related transcripts during viral infection.

• Evolution of antiviral functions: Studies have identified signatures of positive selection in many vertebrate immune proteins , and similar analysis of PRKRIP1 across species could reveal evolutionary adaptation to viral threats.

Research approaches could include:

• Testing PRKRIP1 response to viral infection or immune stimulation in Xenopus cells

• Assessing viral replication in PRKRIP1-depleted versus control conditions

• Identifying PRKRIP1 binding partners during viral infection

How has PRKRIP1 evolved across vertebrate species compared to Xenopus?

Evolutionary analysis of PRKRIP1 across vertebrates provides insights into its conserved functions and species-specific adaptations:

While detailed evolutionary analysis of PRKRIP1 is not provided in the search results, approaches similar to those used for studying RTPs in vertebrates would be valuable. Such analysis might reveal:

• Sequence conservation: Core functional domains likely show higher conservation across species, while regulatory regions might be more divergent.

• Signatures of selection: Analysis could identify if PRKRIP1 has undergone positive selection in certain lineages, potentially indicating adaptation to specific cellular or environmental challenges.

• Duplication events: In tetraploid species like Xenopus laevis, gene duplications might lead to subfunctionalization or neofunctionalization of PRKRIP1 copies.

Comparative analysis would involve alignment of PRKRIP1 sequences across species, phylogenetic analysis, and tests for selection such as dN/dS ratios or McDonald-Kreitman tests.

How do the expression patterns of PRKRIP1 differ between Xenopus laevis and Xenopus tropicalis?

Comparing PRKRIP1 expression between Xenopus laevis and Xenopus tropicalis could reveal important biological differences:

While specific comparative expression data is not provided in the search results, such analysis could reveal:

• Temporal expression differences: The timing of PRKRIP1 expression during development might differ between these related species.

• Tissue-specific patterns: Expression might be more restricted or ubiquitous in one species compared to the other.

• Response to stimuli: Differences in how PRKRIP1 expression changes in response to environmental or developmental cues.

• Allele-specific expression: In X. laevis, which is allotetraploid, different copies of PRKRIP1 might show differential expression patterns.

Research approaches could include RNA-seq analysis across developmental stages and tissues, in situ hybridization, and quantitative PCR. The Normal Table of Xenopus development provides a standardized framework for such comparative developmental studies.

What functional differences exist between PRKRIP1 proteins from different vertebrate species?

Functional differences in PRKRIP1 across vertebrate species likely reflect adaptations to species-specific cellular environments:

While detailed functional comparisons are not provided in the search results, potential differences might include:

• Binding specificity: Differences in RNA or protein binding preferences between species.

• Regulatory mechanisms: Variations in how PRKRIP1 is regulated post-translationally.

• Subcellular localization: Differences in nuclear vs. cytoplasmic distribution.

• Interaction networks: Species-specific interacting partners leading to functional diversification.

• Response to environmental factors: Differential responses to stress, temperature, or pathogens.

Experimental approaches for comparative functional analysis could include:

• Cross-species complementation assays

• In vitro binding and activity assays with recombinant proteins from different species

• Structural studies comparing interaction surfaces

• Heterologous expression in Xenopus oocytes for functional comparisons