Recombinant Xenopus laevis RNA-binding protein 24-A (rbm24-a)

What is the basic structure of RBM24-A in Xenopus laevis?

RBM24 is characterized by a highly conserved RNA-recognition motif (RRM) and contains four exons that encode a protein of approximately 224 amino acids. The protein contains specific GT-rich binding regions that mediate its RNA-binding activity. In Xenopus laevis, RBM24 has a similar structure to its mammalian counterparts, featuring the RRM domain that allows it to recognize specific RNA sequences . The full amino acid sequence includes recognizable functional domains that facilitate its role in RNA processing and stability regulation.

What are the primary cellular functions of RBM24-A?

RBM24-A functions primarily as a splicing regulator and RNA stability modulator. It directly binds to the GT-rich regions in mRNA (particularly at the 3'-UTR) to influence mRNA stability, thereby affecting protein translation . Through this mechanism, RBM24 regulates the stability of various mRNAs including P53, P21, and P63 . Additionally, RBM24 plays critical roles in developmental processes, particularly in neural plate patterning, neurogenesis, neural crest development, and axial muscle segmentation in Xenopus embryos . Its ability to regulate alternative splicing switches also contributes to its importance in embryonic development.

How is RBM24-A expression regulated during development?

RBM24 shows specific spatial and temporal expression patterns during development. In Xenopus, RBM24 is predominantly expressed in the nervous system, and in several cases, its expression is specific to neural tissue . Research indicates that RBM24 is significantly upregulated during myocardial differentiation of embryonic stem cells and plays roles in eye development . The expression is dynamically regulated throughout development, with expression patterns changing during neural plate formation, neurogenesis, and muscle segmentation, suggesting stage-specific functions in embryonic development.

What are the optimal methods for expressing recombinant Xenopus laevis RBM24-A in heterologous systems?

For recombinant expression of Xenopus laevis RBM24-A, several expression systems have been utilized with varying efficiency. The yeast protein expression system has proven to be the most economical and efficient eukaryotic system for both secretion and intracellular expression of RBM24 . When higher fidelity of post-translational modifications is required, mammalian cell systems (particularly HEK-293 cells) provide very high-quality protein that closely resembles the natural protein, though with lower yields and higher costs .

For structural and functional studies, E. coli-based expression systems can produce sufficient quantities of protein, especially when tagged with purification tags such as His-tag. The choice of expression system should be determined by the specific research goals:

• Yeast systems: For economical large-scale production with basic eukaryotic modifications

• Mammalian systems: For studies requiring native-like modifications

• E. coli: For structural studies requiring high protein yields

What techniques are most effective for studying RBM24-A interactions with target mRNAs?

RNA immunoprecipitation (RIP) assay has proven to be highly effective for detecting protein-RNA associations involving RBM24. This technique successfully demonstrated that RBM24 directly binds to PTEN mRNA . To assess the impact of RBM24 on mRNA stability, researchers have effectively employed transcriptional inhibition using actinomycin D followed by qPCR analysis to measure mRNA decay rates .

For mapping specific binding sites, a strategic approach involves cloning GT-rich regions from target mRNA 3'-UTRs into reporter plasmids (such as GFP-expressing vectors), followed by co-transfection with RBM24 expression vectors. This approach successfully identified that RBM24 binds specifically to GT-rich regions at positions 8101-8251 in the 3'-UTR of PTEN mRNA . Additionally, in vitro binding assays with purified recombinant protein and synthetic RNA oligonucleotides can provide quantitative binding parameters.

How can RBM24-A function be effectively modulated in Xenopus embryos for developmental studies?

Two primary approaches have proven effective for modulating RBM24 function in Xenopus embryos:

• Overexpression via mRNA injection: Synthetic mRNA encoding RBM24 can be microinjected into Xenopus embryos at early cleavage stages. This approach has demonstrated that elevated RBM24 levels lead to neural plate mispatterning and abnormal axial muscle segmentation, providing insights into its developmental functions .

• Morpholino-mediated knockdown: Antisense morpholino oligonucleotides designed to block RBM24 translation can be injected into embryos. This approach has revealed that Rbmx (a related RNA-binding protein) is necessary for normal anterior neural plate patterning, neurogenesis, neural crest development, and axial muscle segmentation . Similar approaches can be applied to study RBM24-A specifically.

For spatial control of these manipulations, targeted injections into specific blastomeres allow for region-specific analysis. Combining these approaches with lineage tracers (such as fluorescent dextran) enables tracking of manipulated cells throughout development.

What is the role of RBM24-A in neural development of Xenopus laevis?

RBM24-A plays critical roles in multiple aspects of neural development in Xenopus laevis. Expression cloning screens identified RBM24 among RNA-binding proteins that significantly affect neural plate patterning . Experimental evidence indicates that modulating RBM24 levels through mRNA injection results in neural plate mispatterning, suggesting its importance in early neural development .

Specifically, RBM24 is necessary for normal anterior neural plate patterning, neurogenesis, and neural crest development . The protein's expression pattern is predominantly in the nervous system, in many cases specific to neural tissue, further supporting its specialized neural functions . Its role likely involves regulating alternative splicing of key neural developmental genes and/or controlling mRNA stability of factors essential for neural specification and differentiation. The precise neural gene targets of RBM24-A in Xenopus remain an active area of investigation but likely include factors in the neural determination and differentiation pathways.

How does RBM24 function in cancer models, and can findings from Xenopus studies inform cancer research?

RBM24 demonstrates significant tumor-suppressive functions in colorectal cancer models. In colorectal cancer cells, RBM24 overexpression suppresses cell proliferation, migration, invasion, and increases sensitivity to chemotherapeutic agents such as 5-FU and cisplatin . Mechanistically, RBM24 functions by maintaining PTEN mRNA stability through direct binding to the GT-rich region in its 3'-UTR, thereby prolonging PTEN mRNA half-life and increasing PTEN protein expression . This upregulation of PTEN leads to inhibition of the PI3K-Akt signaling pathway, a key oncogenic pathway in many cancers .

The conservation of RBM24 structure and function between Xenopus and mammals suggests that findings from Xenopus studies can inform cancer research. The developmental roles of RBM24 in Xenopus, particularly in regulating cell proliferation and differentiation, parallel its tumor-suppressive functions. Understanding the molecular mechanisms by which RBM24 regulates RNA processing during embryonic development could provide insights into how its dysregulation contributes to cancer progression. Furthermore, Xenopus embryos could serve as an efficient in vivo system for screening compounds that modulate RBM24 activity, potentially identifying new therapeutic approaches for cancers with altered RBM24 expression.

What phenotypes result from RBM24-A knockdown or overexpression in Xenopus embryos?

Manipulation of RBM24-A levels in Xenopus embryos produces distinct developmental phenotypes:

Overexpression phenotypes:

• Neural plate mispatterning with altered expression of neural markers

• Abnormal axial muscle segmentation affecting somite formation and organization

• Potential disruption of normal embryonic patterning due to altered RNA processing of key developmental factors

Knockdown phenotypes:
Based on studies of related RNA-binding proteins like Rbmx in Xenopus:

• Defects in anterior neural plate patterning

• Impaired neurogenesis with reduced expression of neural differentiation markers

• Abnormal neural crest development affecting migration and differentiation

• Disrupted axial muscle segmentation resulting in irregular somite boundaries

These phenotypes highlight the essential role of RBM24-A in multiple developmental processes and suggest that precise regulation of its expression and activity is crucial for normal embryonic development.

How does RBM24-A specifically recognize its target mRNAs, and what determines binding specificity?

RBM24-A recognizes its target mRNAs through its RNA-recognition motif (RRM), which specifically binds to GT-rich regions in the 3'-UTR of target mRNAs . Studies have demonstrated that RBM24 directly binds to the GU(A)GUGU site on RNA, affecting mRNA maturation or translation . This sequence-specific recognition is critical for its function in regulating mRNA stability.

The binding specificity is determined by:

• The presence of specific GT-rich sequences in target mRNAs

• The structural context of these sequences (accessibility within the mRNA secondary structure)

• Potential cooperative binding with other RNA-binding proteins

Research on PTEN mRNA demonstrated that RBM24 specifically binds to the GT-rich region at positions 8101-8251 in the 3'-UTR, highlighting the precision of this recognition . This specificity allows RBM24 to regulate distinct subsets of mRNAs involved in particular developmental or cellular processes. Further structural studies of the RBM24-RNA complex would provide additional insights into the molecular determinants of this specificity.

What is the relationship between RBM24-A's developmental functions and its role in mRNA stability?

RBM24-A's developmental functions are intricately linked to its ability to regulate mRNA stability. During development, precise temporal and spatial control of gene expression is essential, and post-transcriptional regulation through mRNA stability provides an additional layer of control.

RBM24 binds directly to target mRNAs and enhances their stability, as demonstrated with PTEN mRNA where RBM24 overexpression extended its half-life . This mechanism allows RBM24 to fine-tune the expression of key developmental regulators by modulating how long their mRNAs persist in the cell. In neural development and muscle segmentation, this could enable the creation of expression gradients or sharp boundaries of developmental regulators necessary for proper patterning and differentiation.

Additionally, RBM24 has been shown to regulate alternative splicing switches , which adds another dimension to its developmental functions. By influencing both mRNA processing and stability, RBM24 can orchestrate complex gene expression programs required for proper embryonic development. The specific mRNA targets regulated by RBM24 during Xenopus development likely include key factors in neural specification, patterning, and muscle development pathways.

How does RBM24-A interact with other RNA-binding proteins and regulatory networks?

While direct evidence of RBM24-A interactions with other RNA-binding proteins (RBPs) is limited in the provided search results, several inferences can be made based on its functions:

• Co-regulatory networks: RBM24 likely functions within networks of RBPs that cooperatively regulate shared target mRNAs. In Xenopus, multiple RBPs were identified in the same expression cloning screen that affected neural plate patterning, suggesting they may function in related or parallel pathways .

• Competitive binding: RBM24 may compete with other RBPs for binding to overlapping target sequences, creating complex regulatory dynamics that fine-tune gene expression.

• Integration with signaling pathways: RBM24's regulation of PTEN and subsequent effects on the PI3K-Akt pathway demonstrate its integration with key signaling networks . This suggests RBM24 functions at the intersection of post-transcriptional regulation and signal transduction.

• Splicing complexes: As RBM24 regulates alternative splicing, it likely interacts with core components of the spliceosome and other splicing regulators such as SR proteins and hnRNPs, many of which were also identified in the Xenopus screen .

The full complexity of these interactions remains to be elucidated and represents an important frontier in understanding RBM24 function in development and disease.

What are common challenges in purifying recombinant Xenopus laevis RBM24-A and how can they be addressed?

Purification of recombinant Xenopus laevis RBM24-A presents several challenges that researchers should anticipate:

• Protein solubility issues: RBM24, like many RNA-binding proteins, can form insoluble aggregates during expression. This can be addressed by:

  • Optimizing expression temperature (typically lowering to 16-18°C)

  • Using solubility-enhancing tags (such as SUMO or MBP)

  • Adding RNA during purification to stabilize the protein's native conformation

• Maintaining RNA-binding activity: The functional integrity of the RNA-recognition motif is critical for studies requiring active protein. Consider:

  • Avoiding harsh elution conditions during affinity purification

  • Including reducing agents to prevent oxidation of cysteine residues

  • Verifying activity through RNA-binding assays post-purification

• Expression system selection: Different expression systems yield varying protein quality and quantity. Based on the search results:

  • Yeast systems offer a good balance of yield and post-translational modifications

  • Mammalian systems (HEK-293 cells) provide the highest quality protein with proper folding and modifications, but at higher cost

  • E. coli systems may require optimization of codon usage for Xenopus proteins

• Purification strategy: A multi-step purification approach typically yields the best results:

  • Initial affinity purification using tags (His, GST, or Strep tags appear effective based on available products)

  • Secondary ion-exchange or size-exclusion chromatography for higher purity

How can researchers ensure the functional activity of purified RBM24-A for in vitro studies?

Ensuring the functional activity of purified RBM24-A is critical for meaningful in vitro studies. Several approaches can verify and maintain functional integrity:

• RNA-binding assays: Perform electrophoretic mobility shift assays (EMSA) using synthetic RNA oligonucleotides containing known RBM24 binding motifs (GT-rich sequences) to confirm binding activity.

• Thermal stability assessment: Use differential scanning fluorimetry (DSF) in the presence and absence of target RNA to assess protein stability and proper folding.

• Storage conditions optimization:

  • Include glycerol (typically 10-20%) to prevent freeze-thaw damage

  • Store small aliquots to avoid repeated freeze-thaw cycles

  • Consider flash-freezing in liquid nitrogen for long-term storage

• Functional validation: Before complex experiments, verify that purified RBM24-A can:

  • Stabilize target mRNAs in a cell-free mRNA decay assay

  • Bind specifically to known target sequences in RIP-like in vitro assays

• Quality control metrics: Establish clear criteria for batch-to-batch consistency:

  • Specific activity measurements (RNA binding per μg protein)

  • Purity assessment by SDS-PAGE (>90% purity as a minimum standard)

  • Absence of contaminating nucleases that could degrade target RNAs

What controls are essential when studying RBM24-A's effects on mRNA stability and translation?

• Protein-specific controls:

  • Inactive RBM24 mutant (mutations in the RNA-recognition motif) to confirm specificity

  • Dose-dependent analysis to establish relationship between RBM24 levels and observed effects

  • Related RBPs as specificity controls to ensure effects are specific to RBM24

• mRNA target controls:

  • Reporter constructs with mutated GT-rich regions to confirm binding site specificity

  • Multiple independent target mRNAs to establish breadth of effects

  • Non-target mRNAs (lacking GT-rich regions) as negative controls

• Experimental approach controls:

  • For actinomycin D-based mRNA stability assays: include housekeeping mRNAs with known decay rates

  • For translation studies: measure both protein and mRNA levels to distinguish translational from stability effects

  • Time course experiments to capture the dynamics of RBM24 effects

• System-specific controls:

  • When using RBM24 knockdown approaches, include rescue experiments with RNAi-resistant RBM24 constructs

  • For overexpression studies, ensure expression levels are within physiologically relevant ranges

  • When studying PTEN/PI3K/Akt pathway effects, use pathway-specific inhibitors (SF1670, MK-2206, PI3K-IN-6) as demonstrated in colorectal cancer studies

These controls ensure that observed effects can be confidently attributed to RBM24's specific activity on target mRNAs rather than to experimental artifacts or indirect effects.

What are the key unanswered questions about RBM24-A function in Xenopus development?

Several critical questions about RBM24-A function in Xenopus development remain to be fully addressed:

• Comprehensive target identification: What is the complete repertoire of mRNAs directly regulated by RBM24-A during different stages of Xenopus development? Transcriptome-wide binding site analysis (CLIP-seq) in developing Xenopus embryos would provide valuable insights.

• Developmental timing mechanisms: How does RBM24-A contribute to the precise temporal regulation of developmental processes? Does its activity change at different developmental stages, and what regulates these changes?

• Tissue-specific functions: While RBM24-A is expressed in neural tissues, its specific functions may vary between neural subtypes or regions. What are the tissue-specific roles and targets of RBM24-A in different parts of the developing nervous system?

• Integration with signaling pathways: How does RBM24-A activity intersect with major developmental signaling pathways (Wnt, BMP, FGF, Notch)? Does it regulate components of these pathways at the post-transcriptional level?

• Evolutionary conservation: How conserved are RBM24-A functions between Xenopus and other vertebrates, and what aspects of its regulation have diverged during evolution?

Addressing these questions would significantly advance our understanding of RBM24-A's role in vertebrate development and potentially reveal new principles of post-transcriptional regulation during embryogenesis.

How might comparative studies between Xenopus RBM24-A and mammalian RBM24 advance understanding of RNA regulation?

Comparative studies between Xenopus RBM24-A and mammalian RBM24 offer several promising avenues for advancing our understanding of RNA regulation:

• Conserved regulatory mechanisms: Identifying the core RNA-binding preferences and regulatory mechanisms conserved between species would reveal fundamental aspects of RBM24 function. The conservation of the PTEN regulation mechanism could be examined across species to determine if this represents a universal function of RBM24.

• Species-specific targets: Comparing the mRNA targets of RBM24 across species could reveal how RNA regulation has evolved to accommodate species-specific developmental programs. This might explain differences in developmental timing or tissue organization between Xenopus and mammals.

• Structural insights: Comparative structural analysis of Xenopus and mammalian RBM24 in complex with target RNAs could reveal conserved and divergent aspects of RNA recognition, potentially identifying structural features that could be targeted therapeutically.

• Functional complementation experiments: Testing whether Xenopus RBM24-A can rescue phenotypes in mammalian cells lacking RBM24 (and vice versa) would reveal the degree of functional conservation and potentially identify species-specific cofactors or regulatory mechanisms.

• Developmental context differences: Examining how the developmental context influences RBM24 function in each species could reveal principles about how RNA regulation is integrated into diverse developmental programs.

These comparative approaches would not only advance our understanding of RBM24 biology but also provide broader insights into the evolution and plasticity of post-transcriptional regulatory networks.

What potential therapeutic applications might emerge from detailed understanding of RBM24 function?

Understanding RBM24 function in detail could lead to several promising therapeutic applications:

• Cancer therapeutics: Given RBM24's role as a tumor suppressor in colorectal cancer through PTEN regulation , strategies to restore or enhance RBM24 expression or function could have anti-cancer effects. The observation that RBM24 overexpression increases sensitivity to chemotherapeutic agents like 5-FU and cisplatin suggests that RBM24-targeting approaches could enhance conventional chemotherapy efficacy.

• RNA-based therapeutics: Detailed knowledge of how RBM24 stabilizes specific mRNAs could inform the design of synthetic RNA stabilization approaches. Mimicking RBM24's binding to therapeutic mRNAs could enhance their stability and translation efficiency.

• Developmental disorder treatments: As RBM24 plays critical roles in neural and muscle development , understanding its function could provide insights into developmental disorders affecting these tissues. Modulating RBM24 activity might help address certain congenital neural or muscular abnormalities.

• Regenerative medicine applications: If RBM24's developmental functions in neural and muscle tissues can be harnessed in adult contexts, it might have applications in promoting tissue regeneration after injury or in degenerative conditions.

• Diagnostic biomarkers: The correlation between RBM24 expression and clinical outcomes in colorectal cancer suggests potential applications as a prognostic or predictive biomarker for certain cancers.

The translational potential of RBM24 research underscores the importance of continuing to investigate its molecular functions and regulatory networks in both developmental and disease contexts.