Recombinant Oncorhynchus mykiss Piwi-like protein 2 (piwil2), partial

What is piwil2 and what is its fundamental role in Oncorhynchus mykiss?

Piwil2 (Piwi-like protein 2) in Oncorhynchus mykiss (rainbow trout) is a member of the evolutionary conserved Piwi protein family. The primary function of piwil2 appears to be repression of transposable elements, which is critical for maintaining germline cell fate and genome integrity. Research in related fish species like Nile tilapia demonstrates that piwil2 is a gonad-specific and maternally deposited gene in fish eggs, suggesting its importance in early development . The protein contains distinctive functional domains including the PAZ domain (an RNA binding motif) and the PIWI domain, which has a structure similar to the RNase H catalytic domain and functions as a catalytic engine in RNA-induced silencing complexes (RISC) .

What specific domains characterize piwil2 protein and what are their functional significance?

Piwil2 contains two principal functional domains:

• PAZ domain: Functions as an RNA-binding motif that interacts with small RNAs

• PIWI domain: Structurally similar to RNase H catalytic domain and acts as the catalytic core in RNA-induced silencing complexes (RISC)

How conserved is piwil2 across different fish species compared to mammals?

Piwil2 appears to be functionally conserved between fish and mammals, though with some tissue-specific differences in expression patterns. Research indicates that piwil2, along with other genes like nanos2, nanos3, pou2, and plzf, is preferentially expressed in undifferentiated spermatogonia in fish, similar to their expression in mammalian systems . This conservation suggests fundamental roles in germline development across vertebrate species.

In mammals, particularly mice, Piwil2 (also called Mili) functions extend beyond germ cells, as it has been shown to be essential for proper neurogenesis in the postnatal hippocampus . This neural function hasn't been extensively characterized in fish models, representing a potential area for comparative research.

What are the optimal storage and reconstitution protocols for recombinant piwil2?

For optimal results when working with recombinant Oncorhynchus mykiss piwil2, follow these evidence-based storage and reconstitution protocols:

Storage Recommendations:

• Store at -20°C for regular use

• For extended storage, conserve at -20°C or -80°C

• Avoid repeated freeze-thaw cycles which may compromise protein integrity

• Working aliquots can be stored at 4°C for up to one week

Reconstitution Protocol:

• Centrifuge the vial briefly before opening to bring contents to the bottom

• Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (50% is recommended) to enhance stability

• Create multiple aliquots to prevent repeated freeze-thaw cycles

Shelf Life Information:

• Liquid form: 6 months at -20°C/-80°C

• Lyophilized form: 12 months at -20°C/-80°C

What factors influence the stability of recombinant piwil2 in experimental settings?

Several factors can affect the stability and experimental utility of recombinant piwil2:

• Temperature conditions: As a protein derived from a poikilothermic species (rainbow trout), temperature optimization is critical. Research on CRISPR/Cas9 applications in fish species highlights that standard laboratory temperatures may be suboptimal for proteins from cold-water species like rainbow trout .

• Buffer composition: The specific buffer ingredients significantly impact protein stability. While detailed buffer optimization data for piwil2 specifically is limited, the recommended use of glycerol (5-50%) in storage buffers suggests its importance for maintaining protein structure .

• Freeze-thaw cycles: Repeated freezing and thawing is explicitly not recommended for piwil2, as this can lead to protein denaturation and activity loss .

• Protein concentration: Working at appropriate concentrations (0.1-1.0 mg/mL is recommended) helps maintain protein stability and functionality .

• pH and salt concentration: While specific data for rainbow trout piwil2 isn't provided in the source materials, these are generally critical factors for maintaining proper protein folding and activity.

What methodologies have proven most effective for studying piwil2 function in fish models?

Research demonstrates several effective methodologies for studying piwil2 function in fish models:

CRISPR/Cas9 Gene Editing:

• Successfully used to target the PIWI domain of piwil2 in Nile tilapia

• Typically involves injection of Cas9 mRNA (500 ng/μL) and sgRNA (150 ng/μL) into embryos at the 1-cell stage

• Has achieved mutation frequencies of 95.8 ± 4.3% with survival rates to 3 days post-fertilization (37.8 ± 18.6%) comparable to uninjected controls (42.5 ± 10.8%)

Histological Analysis:

• Used to identify primordial germ cells (PGCs) based on location and morphological features

• Essential for phenotypic assessment of piwil2 knockout effects

3. Mutant Screening Methods:
Multiple techniques have been used to verify and characterize CRISPR/Cas9-induced mutations:

Studies suggest that the complex mosaicism and wide indel spectrum produced in F0 CRISPR knockouts make comprehensive analysis challenging, with NGS and fragment analysis providing the most detailed information .

How does piwil2 knockdown affect primordial germ cell development in fish models?

CRISPR/Cas9-mediated knockout of piwil2 in Nile tilapia has revealed significant impacts on primordial germ cell (PGC) development:

• Reduction in PGC numbers: 54% of piwil2 knockout larvae showed either no PGCs or significantly reduced PGC populations compared to control fish .

• Developmental timing: Effects were observable at early larval stages (pre-first feeding), indicating piwil2's importance in early germ cell development rather than just in later gametogenesis .

• Cellular mechanisms: Research suggests piwil2 is essential for PGC survival rather than just specification or migration, consistent with its known role in transposon repression and genome integrity maintenance .

These findings provide strong evidence for the functional importance of piwil2 in maintaining primordial germ cell populations in fish, suggesting potential applications in reproductive technologies and sterility induction for aquaculture .

What experimental approaches can effectively evaluate piwil2's interaction with transposable elements?

While the search results don't provide specific protocols for assessing piwil2-transposon interactions in rainbow trout, effective experimental approaches can be inferred from related research:

• Small RNA sequencing: Analysis of piwil2-associated small RNAs (piRNAs) to identify those targeting transposable elements. Studies in mice have shown that directionality analysis of small RNA sequencing data can reveal strand bias, where most piRNAs arise unidirectionally, though some piRNAs are bidirectional .

• RNA immunoprecipitation (RIP): Immunoprecipitating piwil2 complexes followed by RNA sequencing to identify bound transposon RNAs.

• Transposon mobilization assays: Comparing transposon activity in wild-type versus piwil2-depleted cells to quantify the protein's repressive effects.

• Expression analysis of transposable elements: RT-qPCR or RNA-seq to measure expression levels of specific transposable elements following piwil2 knockdown or knockout.

• Chromatin immunoprecipitation (ChIP): To identify potential interactions between piwil2 and transposon-associated chromatin.

These approaches would need to be optimized specifically for rainbow trout systems, considering the temperature and other physiological differences of this poikilothermic species.

How does piwil2 function compare between germ cells and neural tissues?

Research reveals interesting differences in piwil2 function between germline and neural tissues:

Germline Function:

• In fish, piwil2 is gonad-specific and maternally deposited in eggs

• Essential for primordial germ cell survival during early development

• Primary role involves transposon repression to maintain genomic integrity in germline cells

• In mammalian systems, it's associated with early stages of gametogenesis

Neural Function (primarily studied in mammals):

• Piwil2 (Mili) and associated piRNAs are dynamically expressed during neurogenesis in the postnatal mouse hippocampus

• Essential for proper neural progenitor cell (NPC) differentiation toward neural fate

• Prevents cellular senescence in adult neural progenitor cells

• Depletion leads to generation of reactive glia rather than neurons

• Regulates transcripts bearing sequences complementary or homologous to piRNAs, including repetitive elements and mRNAs essential for neurogenesis

While neural functions have been characterized in mammalian systems, there's limited data on piwil2's role in fish neural tissues, representing a significant research opportunity for comparative studies.

What is known about the mechanism through which piwil2 regulates cellular processes beyond transposon silencing?

Recent research has uncovered several mechanisms through which piwil2 regulates cellular processes beyond its canonical role in transposon silencing:

• Protein-Protein Interactions: In esophageal squamous cell carcinoma (ESCC), PIWIL2 directly interacts with IKK (a major regulator of the canonical NF-κB pathway) to enhance IKK phosphorylation, leading to a cascade that inhibits apoptosis .

• Competitive Inhibition: PIWIL2 has been shown to competitively inhibit the binding of IKK to TSC1, thereby deactivating the mTORC1 pathway, which suppresses ULK1 phosphorylation and initiates autophagy .

• Transcriptional Regulation: In neural progenitor cells, Piwil2 (Mili) regulates transcripts that contain sequences complementary or homologous to piRNAs, affecting expression of genes essential for proper neurogenesis .

• Cellular Fitness Maintenance: Depletion of Piwil2 in adult neural progenitor cells induces cellular senescence, suggesting a role in maintaining cellular fitness beyond germline cells .

These diverse mechanisms highlight piwil2's multifunctional nature across different cellular contexts and suggest potential therapeutic applications in areas like cancer treatment and neurodegenerative disorders.

How do different mutant screening methods compare when studying piwil2 knockout models?

When studying piwil2 knockout models, particularly in F0 generations created through CRISPR/Cas9, various mutant screening methods show different strengths and limitations:

Research on piwil2 knockout in Nile tilapia revealed complex mosaicism and wide indel diversity that made linking phenotypes to genotypes in F0 generations particularly challenging. NGS and fragment analysis provided the most comprehensive information about the genetic changes .

For future piwil2 studies, recommendations include:

• Using multiple complementary methods for comprehensive mutation characterization

• Considering techniques to reduce mosaicism when using CRISPR/Cas9 to facilitate direct functional analysis in F0 generations

• Progressing to F1 or later generations for cleaner genotype-phenotype correlations

What are the promising research directions for piwil2 in fisheries and aquaculture applications?

Several promising research directions for piwil2 in fisheries and aquaculture include:

• Sterility Induction: piwil2 has been identified as a potential target for inducing sterility in farmed fish through gene knockout. Research in Nile tilapia has already demonstrated that piwil2 disruption leads to significant reduction in primordial germ cells . This application could help address concerns about genetic contamination from farmed fish escaping into wild populations.

• Reproductive Technology Optimization: Better understanding of piwil2's role in germline development could lead to improved reproductive management techniques for commercially important species like rainbow trout.

• Development of Non-GMO Sterility Methods: Insights from piwil2 function could inform development of non-genetic methods to induce temporary sterility in farmed fish.

• Biomarkers for Reproductive Health: piwil2 expression patterns could potentially serve as biomarkers for reproductive health assessment in wild and farmed fish populations.

• Comparative Studies Across Commercially Important Species: Expanding piwil2 research to other commercially relevant fish species beyond rainbow trout and Nile tilapia.

What contradictions or knowledge gaps exist in current piwil2 research?

Several significant knowledge gaps and potential contradictions exist in current piwil2 research:

• Species-Specific Variations: While piwil2 function has been studied in Nile tilapia and some data exists for rainbow trout, there's limited comparative analysis across fish species to understand species-specific variations in function and regulation.

• Non-Germline Functions in Fish: There is substantial evidence for Piwil2's role in neurogenesis in mice , but similar studies in fish are lacking. This represents a significant knowledge gap in understanding potential conservation of non-germline functions.

• Molecular Interactions: While some protein interaction partners have been identified in cancer contexts (e.g., IKK in ESCC) , the complete interactome of piwil2 in normal fish tissues remains poorly characterized.

• Temporal Dynamics: The precise timing and regulation of piwil2 expression throughout the life cycle of rainbow trout is not well documented in the literature.

• Methodological Challenges: Current research highlights difficulties in analyzing F0 CRISPR/Cas9 knockout fish due to mosaicism , suggesting a need for improved methodological approaches.

• Temperature Effects: As rainbow trout is a cold-water species, the temperature dependency of recombinant piwil2 function in experimental settings represents another important area for investigation.

Addressing these knowledge gaps would significantly advance our understanding of piwil2 biology and its potential applications in fisheries, aquaculture, and possibly biomedical research.