Recombinant Xenopus tropicalis RNA-binding protein 5 (rbm5), partial

What is RNA-binding protein 5 (RBM5) and what are its key structural features in Xenopus tropicalis?

RBM5 is a multi-domain RNA-binding protein that functions as a regulator of alternative splicing. In Xenopus tropicalis, RBM5 contains several characteristic domains:

• Two RNA Recognition Motif (RRM) domains

• Two zinc finger (ZF) domains (specifically RanBP ZF type)

• An OCRE (OCtamer REpeat of aromatic residues) domain

The domain architecture of Xenopus tropicalis RBM5 includes:

The RRM domains harbor two consensus motifs involved in RNA interaction: RNP1 ([RK]-G-[FY]-[GA]-[FY]-[ILV]-X-[FY]) and RNP2 ([ILV]-[FY]-[ILV]-X-N-L). Notably, both RBM5 RRM domains are non-canonical as they lack the consensus aromatic residue in RNP2, which impacts their RNA recognition capabilities .

Why is Xenopus tropicalis a suitable model system for studying RBM5 function?

Xenopus tropicalis has emerged as a powerful amphibian genetic model system for several reasons when studying proteins like RBM5:

• It offers experimental advantages similar to its larger cousin, Xenopus laevis, while being more amenable to genetic manipulation

• High efficiency of genome editing techniques: TALENs targeting genes in X. tropicalis can achieve bi-allelic mutations at very high frequency (>90%) in F0 animals

• Mutations are efficiently transmitted to F1 progeny

• The transparent nature of embryonic development allows for direct visualization of developmental processes

• Its genome has been sequenced and annotated, facilitating genomic and transcriptomic analyses

• It has a shorter generation time compared to X. laevis, making genetic studies more feasible

These advantages make X. tropicalis particularly useful for studying the roles of RNA-binding proteins like RBM5 in development, RNA processing, and disease models.

What are the optimal storage and handling conditions for recombinant Xenopus tropicalis RBM5?

For optimal stability and activity of recombinant Xenopus tropicalis RBM5 protein:

Storage Conditions:

• Store at -20°C for regular use

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

• Avoid repeated freezing and thawing (not recommended)

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

Reconstitution Protocol:

• Briefly centrifuge the vial prior to opening to bring contents to the bottom

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

• Add 5-50% of glycerol (final concentration) and aliquot for long-term storage

• Standard final concentration of glycerol is 50%

Shelf Life:

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

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

Note that shelf life depends on multiple factors including storage state, buffer ingredients, storage temperature, and the intrinsic stability of the protein itself .

What purification and quality control methods are recommended for recombinant Xenopus tropicalis RBM5?

For optimal purification and quality control of recombinant X. tropicalis RBM5:

Purification Methods:

• Recombinant expression in yeast systems has been successful

• Affinity chromatography using His-tags is commonly employed for initial purification

• Ion exchange chromatography can be used for further purification

• Size exclusion chromatography helps ensure homogeneity

Quality Control Standards:

• Purity should be >85% as determined by SDS-PAGE

• Western blotting with specific antibodies confirms identity

• Mass spectrometry can verify molecular weight and post-translational modifications

• Functional activity assays such as RNA-binding assays validate biological activity

For RNA-binding studies specifically, it's essential to confirm that the purified protein maintains its native folding and RNA-binding capacity. This can be assessed through:

• Circular dichroism spectroscopy to verify secondary structure

• NMR spectroscopy to evaluate tertiary structure

• Electrophoretic mobility shift assays (EMSA) to test RNA-binding capability

What is known about RBM5's role in Xenopus development compared to its role in mammals?

RBM5's developmental roles exhibit both conserved and divergent patterns between Xenopus and mammals:

Conserved Functions:

• Regulation of alternative splicing of target mRNAs

• Interaction with components of the spliceosome

• Recognition of similar RNA motifs through conserved RNA-binding domains

Xenopus-Specific Aspects:

• While comprehensive studies on RBM5's role in Xenopus development are still emerging, its presence in early developmental stages suggests involvement in embryonic patterning

• TALENs-based studies in X. tropicalis provide tools to investigate its developmental functions

Mammalian Comparative Data:

• In mammals, RBM5 is critical for spermatid differentiation and male fertility

• RBM5 localizes to somatic and germ cells in adult mouse testis

• The R263P mutation in mice affects pre-mRNA splicing and results in shifts in isoform ratios of target genes

• Mouse studies revealed that RBM5 is required for spermatid head shaping, acrosome formation, and tail development

Research indicates that RBM5's function in splicing regulation appears to be evolutionarily conserved, though the specific mRNA targets and developmental processes affected may vary between species. Studying these differences can provide insights into the evolution of post-transcriptional regulatory networks.

How does RBM5 contribute to splicing regulation and what are its known target genes in Xenopus and other model organisms?

RBM5 is an integral component of the splicing machinery that regulates alternative splicing through several mechanisms:

Splicing Regulatory Mechanisms:

• RBM5 is a component of the U2 snRNP involved in branch site recognition

• It interacts with the spliceosome through its OCRE domain, which binds to the proline-rich C-terminal tails of SmN/B/B' proteins

• It can inhibit the transition from pre-spliceosomal complex A to spliceosomal complex B

• The C-terminal region of RBM5 is necessary but not sufficient for inhibition of complex B formation

• RBM5 can also interact with U2AF65, potentially influencing 3' splice site recognition

Known Target Genes:

While specific Xenopus tropicalis targets are still being investigated, the identification of conserved RNA recognition motifs suggests potential conservation of some target genes. Studies in mammals indicate that RBM5 primarily functions as a splicing repressor through interactions with the branch site recognition machinery .

What are effective techniques for studying RBM5-RNA interactions in Xenopus tropicalis?

Multiple complementary approaches can be employed to study RBM5-RNA interactions in Xenopus tropicalis:

In Vitro Methods:

• RNA Electrophoretic Mobility Shift Assay (EMSA): Useful for determining binding affinities between purified recombinant RBM5 and synthetic RNA oligonucleotides

• Isothermal Titration Calorimetry (ITC): Provides thermodynamic parameters of RBM5-RNA interactions, revealing binding affinity, stoichiometry, and enthalpy

• Surface Plasmon Resonance (SPR): Enables real-time monitoring of RBM5-RNA association and dissociation kinetics

• NMR Spectroscopy: Reveals structural details of RBM5-RNA complexes at atomic resolution, as demonstrated in structural studies of RBM5 domains

In Vivo/Cell-Based Methods:

• RNA Immunoprecipitation (RIP): Identifies endogenous RNAs associated with RBM5 in Xenopus cells or tissues

• Cross-Linking Immunoprecipitation (CLIP): More precisely maps RBM5 binding sites on target RNAs

• RNA Pull-Down: Identifies proteins associated with specific RNA sequences, useful for confirming direct RBM5 interactions

• Bimolecular Fluorescence Complementation (BiFC): Visualizes RBM5-RNA interactions in living cells

Computational and High-Throughput Approaches:

• NLP-Based Methods: Novel computational approaches using natural language processing to identify RNA-binding motifs

• Multiple Instance Learning (MIL): Computational framework that can identify precise RNA contexts necessary for RBP binding events

• Consensus-Based Motif Discovery: Algorithms that rely on enrichment, similarity, and co-occurrence of k-mers to discover RNA motifs

The RBM5 RRM2 domain has been shown to preferentially bind to both CU-rich and GA-rich sequences with affinity in the 10^-5 molar range, providing starting points for designing RNA targets for interaction studies .

How can CRISPR-Cas9 or TALENs approaches be optimized for studying RBM5 function in Xenopus tropicalis?

Genome editing using CRISPR-Cas9 or TALENs can be effectively optimized for studying RBM5 function in Xenopus tropicalis:

TALENs Approach Optimization:

• Target the first exon of RBM5 for efficient gene disruption

• Inject mRNA encoding TALENs into two-cell-stage embryos

• TALENs have shown >90% efficiency in generating bi-allelic mutations in X. tropicalis

• Screen for mutations using restriction fragment length polymorphism (RFLP) or T7 endonuclease assays

• Establish F1 lines by breeding mosaic F0 animals to wild-type partners

CRISPR-Cas9 Optimization:

• Design sgRNAs targeting early exons, particularly those encoding critical RNA-binding domains

• Multiple sgRNAs can be used simultaneously to ensure efficient knockout

• Ribonucleoprotein (RNP) delivery (pre-assembled Cas9 protein with sgRNA) rather than mRNA injection can reduce off-target effects

• For domain-specific studies, design precise edits that disrupt specific domains (RRM, ZnF, or OCRE)

Domain-Specific Targeting Strategy:

Validation Methods:

• RT-PCR and western blotting to confirm knockout at RNA and protein levels

• RNA-Seq to identify global changes in alternative splicing

• Rescue experiments with wild-type or domain mutant variants to confirm specificity

The high efficiency of TALENs in X. tropicalis makes this an especially attractive approach for generating stable RBM5 mutant lines.

How does the OCRE domain of RBM5 contribute to spliceosome interactions, and how might this differ in Xenopus compared to mammals?

The OCRE domain plays a crucial role in connecting RBM5 to the core spliceosome machinery:

Structure and Function of the OCRE Domain:

• The RBM5 OCRE domain adopts a unique β-sheet fold

• It directly binds to the proline-rich C-terminal tail of the essential snRNP core proteins SmN/B/B'

• NMR structure reveals that OCRE specifically recognizes poly-proline helical motifs in SmN/B/B'

• Three tyrosine residues (Tyr479, Tyr488, and Tyr495) play crucial roles in recognizing proline-rich regions

• This interaction is critical for RBM5's function in alternative splicing regulation, particularly of FAS/CD95

Functional Significance:

• The OCRE domain is necessary but not sufficient for inhibition of spliceosomal complex B formation

• It mediates interactions with components of the U4/U6.U5 tri-snRNP

• Mutation of conserved aromatic residues impairs binding to Sm proteins in vitro and compromises RBM5-mediated alternative splicing regulation

Potential Xenopus-Mammalian Differences:
While the OCRE domain structure is likely conserved between Xenopus and mammals based on sequence conservation, several aspects may differ:

• The protein sequence of SmN/B/B' C-terminal tails in Xenopus may contain differences that affect binding affinity or specificity

• The complement of spliceosomal proteins expressed in Xenopus tissues may differ from mammals

• The regulatory importance of this interaction may vary between species and developmental contexts

Experimental approaches to investigate these differences could include:

• Comparative binding assays between Xenopus and mammalian OCRE domains with their respective SmN/B/B' peptides

• Structural studies of the Xenopus OCRE-SmN/B/B' complex

• Domain swap experiments to test functional conservation

What are the latest approaches for identifying novel RBM5 RNA targets in Xenopus tropicalis?

Cutting-edge approaches for identifying and validating RBM5 RNA targets in Xenopus tropicalis combine high-throughput techniques with computational analysis:

Enhanced CLIP-Based Approaches:

• iCLIP/eCLIP: Individual-nucleotide resolution CLIP or enhanced CLIP provides single-nucleotide resolution of RBM5 binding sites

• CLIP-seq with Xenopus-specific adaptations: Optimized crosslinking conditions for amphibian tissues and embryos

• Fractionation-assisted CLIP: Isolates RBM5-associated RNAs from specific cellular compartments

RNA-Centric Methods:

• RNA Antisense Purification (RAP): Identifies proteins associated with specific RNAs of interest

• RNA Pull-Down coupled with Mass Spectrometry: Validates direct RBM5 interactions with specific RNA sequences

Novel Computational Approaches:

• NLP-Based Decomposition Method: Deconstructs sequences into entities consisting of central target k-mers and their contexts

• Multiple Instance Learning (MIL) Framework: Treats every k-mer as a candidate target for RBP binding

• Consensus-Based Motif Discovery Algorithm: Uses three key properties of k-mers (enrichment, similarity, co-occurrence) to discover motifs

Integrative Methods:

• RBM5-Specific RNP-seq: Isolates endogenous RBM5-containing ribonucleoprotein complexes and sequences associated RNAs

• Branch Site Mapping: Identifies branch sites bound by RBM5-containing U2 snRNPs

• Comparative Transcriptomics: Compares wild-type and RBM5-deficient Xenopus embryos to identify mis-spliced or mis-regulated transcripts

A recent study demonstrated an NLP-based method that identified 11 round spermatid-expressed mRNAs as putative RBM5 targets in mouse testis. Similar approaches could be adapted for Xenopus studies. Additionally, research has shown that RBM5 is a component of the U2 snRNP that binds branch sites across many exons, which provides a new angle for identifying RBM5-regulated splicing events in Xenopus .

How can researchers resolve contradictory findings about RBM5's role as both a tumor suppressor and essential gene?

The apparent contradiction regarding RBM5's dual roles as both a tumor suppressor and an essential gene can be resolved through several experimental strategies:

Mechanistic Investigation Approaches:

• Context-Dependent Analyses:

  • Compare RBM5 function across different cellular contexts (cancer vs. normal cells, different developmental stages)

  • Analyze tissue-specific conditional knockout models

  • Example: RBM5 knockout significantly impairs acute myeloid leukemia (AML) survival while having minimal effects on normal human myeloid differentiation

• Target Gene Profiling:

  • Identify differentially regulated targets in normal vs. cancer contexts

  • Example: HOXA9/FLT3 axis was identified as a dominant downstream target of RBM5 in leukemia cells

• Domain-Specific Studies:

  • Use domain dropout CRISPR screens to identify which domains are essential in different contexts

  • A C2H2 DNA-binding domain was found to be required for RBM5 function in AML

Experimental Design Strategies:

• Cell Type-Specific Approaches:

  Cell/Tissue Type	RBM5 Effect	Experimental System
  AML cell lines (MOLM13, THP1, OCIAML2)	Essential for survival	CRISPR-Cas9 knockout shows strong growth inhibition
  Other blood cell lines (U937, HEL, TF1)	Mild effect	CRISPR-Cas9 knockout shows minimal growth inhibition
  Normal CD34+ HSPCs	Not required	No effect on myeloid differentiation or colony formation
  Male germ cells	Essential for fertility	Mutation leads to complete block of spermatid differentiation
  Lung cancer cells	Tumor suppressor	Loss promotes tumor growth

• Rescue Experiments:

  • Use sgRNA-resistant RBM5 cDNA to confirm specificity of knockout effects

  • Domain-specific mutants can identify which functions are essential in which contexts

• Temporal Control:

  • Inducible knockout/knockdown systems

  • Developmental stage-specific manipulations in Xenopus

A comprehensive approach would combine these strategies to elucidate how RBM5's functions differ between contexts. For example, in leukemia, RBM5 protein degradation was found to acutely decrease HOXA9 transcription, suggesting a direct link to an oncogenic pathway in that context. Meanwhile, in other tissues, RBM5's splicing regulatory functions may predominate and affect different sets of target genes .

What are emerging areas of RBM5 research that Xenopus tropicalis could help address?

Xenopus tropicalis offers unique advantages for addressing several emerging areas in RBM5 research:

Developmental Role of RBM5 in Neurogenesis:

• X. tropicalis transparent embryos allow real-time visualization of neural development

• Recent findings that RBM5 levels increase after CNS trauma and promote neuronal death in vitro warrant investigation in developmental contexts

• Techniques for neural-specific gene manipulation in Xenopus can help dissect RBM5's role in normal neural development versus its pathological roles after injury

Evolutionary Conservation of Splicing Networks:

• As an amphibian model, X. tropicalis provides an evolutionary perspective between fish and mammals

• Comparative studies of RBM5 targets across species could reveal conserved core functions versus species-specific adaptations

• The accessibility of different developmental stages in large numbers facilitates stage-specific transcriptomic analyses

Role in Cellular Stress Responses:

• X. tropicalis embryos can be easily manipulated to induce various stressors

• Investigation of RBM5's role in mediating splicing changes during stress responses

• Potential for discovering novel environmental response mechanisms

Novel Target Discovery Through Multi-Omics Approaches:

• High-throughput sequencing of X. tropicalis embryos at different developmental stages

• Integration of transcriptomics, proteomics, and functional genomics data

• Application of novel NLP-based computational methods for identifying RNA binding protein motifs

Interplay Between RBM5 and Other RBPs:

• Investigation of functional relationships between RBM5 and other RBPs in X. tropicalis

• Recent findings that RBM5 and RBM10 cross-regulate each other can be further explored

• Potential for discovering novel RBP networks specific to amphibian development

The X. tropicalis model offers particular advantages for these investigations due to the ability to generate large numbers of synchronously developing embryos, amenability to genome editing, and conservation of core developmental pathways with mammals.

What methodological considerations are important when designing experiments to investigate RBM5's role in RNA processing during Xenopus development?

When designing experiments to investigate RBM5's role in RNA processing during Xenopus development, several methodological considerations are crucial:

Developmental Timing and Stage-Specific Analysis:

• RBM5 function may vary across developmental stages

• Design time-course experiments spanning key developmental transitions

• Use staged embryo collections to capture dynamic changes in RBM5 expression and activity

• Consider maternal versus zygotic contributions to RBM5 function

Spatial Resolution Approaches:

• Combine whole-embryo analyses with tissue-specific investigations

• Use microdissection techniques to isolate specific tissues

• Consider single-cell RNA-seq to capture cellular heterogeneity

• Implement spatial transcriptomics methods for positional information

Genetic Manipulation Strategies:

RNA Processing Analysis Methods:

• Implement RNA-seq with sufficient depth to detect alternative splicing events

• Consider long-read sequencing to identify complex isoforms

• Use specialized splicing-sensitive analytical pipelines

• Validate key events with RT-PCR and isoform-specific qPCR

Functional Validation Approaches:

• Rescue experiments with wild-type vs. mutant RBM5

• Cross-species rescue with mammalian RBM5 to test functional conservation

• Target-specific rescue by manipulating downstream splicing events

• Combined approach using biochemical, genetic, and computational methods

Controls and Reproducibility Considerations:

• Include multiple biological replicates across different egg clutches

• Use multiple targeting strategies to confirm specificity

• Implement rescue experiments to validate phenotypes

• Consider potential compensatory mechanisms by related RBPs