Recombinant Pongo abelii RNA-binding protein 48 (RBM48)

What is RNA-binding protein 48 (RBM48) and what is its primary function?

RNA-binding protein 48 (RBM48) is a conserved U12 splicing factor that contains an N-terminal RNA recognition motif (RRM) and a C-terminal RS-rich motif, a domain structure common for SR proteins involved in pre-mRNA splicing . The primary function of RBM48 is to facilitate the splicing of rare U12-type introns by the minor spliceosome. Research in maize has demonstrated that RBM48 functions to promote cell differentiation and repress cell proliferation . The protein is coselected with other U12 splicing factors, particularly zinc finger CCCH-type, RNA binding motif, and Ser/Arg rich 2/Rough endosperm 3 (RGH3), suggesting evolutionary conservation of its function in U12 splicing across eukaryotic lineages that maintain the minor spliceosome .

How conserved is RBM48 across different species?

RBM48 is highly conserved across eukaryotic species that maintain the minor spliceosome. Phylogenetic analyses indicate that the RRM domain of RBM48 is found in 344 eukaryotic species, with 89% of species having an RBM48 domain also containing a RGH3/ZRSR2 RRM domain . This suggests co-evolution of these U12 splicing factors. Notably, RBM48 is absent in model organisms that have lost minor intron genes (MIGs) and the U12 splicing machinery, including algae, nematodes, and slime molds . The conservation pattern of RBM48 across multiple eukaryotic kingdoms strongly suggests that its function in U12 splicing is fundamental to organisms that maintain the minor spliceosome, including primates like Pongo abelii.

What protein-protein interactions are known for RBM48?

RBM48 participates in several key protein-protein interactions that support its role in RNA splicing. Studies have demonstrated interactions between RBM48, RGH3, and U2 Auxiliary Factor (U2AF) subunits, suggesting that major and minor spliceosome factors required for intron recognition form complexes with RBM48 . Additionally, human RBM48 interacts with armadillo repeat containing 7 (ARMC7), and this protein-protein interaction is conserved in maize, suggesting evolutionary conservation of this interaction . These interactions provide insight into how RBM48 may function within the splicing machinery across different species, including potentially in Pongo abelii.

What are the structural features of RBM48?

RBM48 contains two primary structural domains that are critical to its function. The N-terminal region contains an RNA Recognition Motif (RRM) that is specific to the RBM48 protein family and is responsible for RNA binding . The C-terminal region contains a 30 amino acid RS-rich motif, which is characterized by arginine-serine dipeptide repeats . This domain structure is common for SR proteins involved in pre-mRNA splicing and facilitates both RNA recognition and protein-protein interactions essential for spliceosome assembly and function . These structural features are likely conserved in the Pongo abelii ortholog, given the high conservation of RBM48 across species that maintain the U12 spliceosome.

What experimental approaches are most effective for studying RBM48's role in U12 splicing in Pongo abelii?

To study RBM48's role in U12 splicing in Pongo abelii, researchers should employ a multi-faceted approach combining biochemical, genetic, and computational methods:

• RNA-protein crosslinking and immunoprecipitation (CLIP): Utilize UV crosslinking followed by immunoprecipitation with anti-RBM48 antibodies to identify direct RNA targets in Pongo abelii cells . This method would reveal the U12 introns that RBM48 directly interacts with.

• RNA-seq analysis: Compare transcriptomes with and without RBM48 knockdown/knockout to identify affected U12 introns. Calculate Percent Spliced Out (PSO) values to quantify splicing efficiency changes, similar to methods used in maize studies where U12-type introns showed significant retention in rbm48 mutants .

• Co-immunoprecipitation (Co-IP): Determine if Pongo abelii RBM48 interacts with predicted partners like RGH3/ZRSR2, U2AF subunits, and ARMC7 as observed in other species .

• Recombinant protein expression and purification: Express the Pongo abelii RBM48 in systems like baculovirus for in vitro binding and functional studies .

• Domain mutation analysis: Create targeted mutations in the RRM and RS-rich domains to determine their specific contributions to RNA binding and protein interactions.

For all these approaches, comparing results with RBM48 data from other primates would provide evolutionary context for Pongo abelii-specific findings.

How can researchers distinguish between U12-dependent and U12-independent functions of RBM48?

Distinguishing between U12-dependent and potential U12-independent functions of RBM48 requires strategic experimental design:

• Comprehensive splicing analysis: Perform deep RNA-seq after RBM48 depletion and analyze both U12 and U2 intron splicing efficiency. In maize studies, rbm48 mutants showed retention of >50% of U12-type introns, while U2-type introns were minimally affected .

• U12 splice site mutation studies: Create reporter constructs with wild-type and mutated U12 splice sites, then assess whether RBM48 still associates with or affects processing of transcripts with mutated U12 sites.

• Cellular compartmentalization analysis: Use cell fractionation followed by western blotting to determine if RBM48 localizes to cellular compartments lacking U12 splicing activity, which would suggest additional functions.

• Proteomics after RBM48 depletion: Compare proteins affected by RBM48 knockdown with those affected by depletion of other U12-specific factors. Proteins uniquely affected by RBM48 depletion might indicate U12-independent functions.

• Rescue experiments: Test whether U12-specific phenotypes can be rescued by RBM48 mutants lacking domains potentially involved in U12-independent functions.

This multi-faceted approach would help identify any functions of Pongo abelii RBM48 beyond its established role in U12 splicing.

What are the implications of RBM48 dysfunction for cellular differentiation based on comparative analysis with other species?

Based on studies in maize, RBM48 dysfunction has profound implications for cellular differentiation that may have parallels in Pongo abelii:

• Cell differentiation defects: In maize, rbm48 mutations disrupt endosperm cellular development, with cells failing to express cell-type specific markers in the correct domains . This suggests RBM48 is essential for proper cellular differentiation programs.

• Proliferation vs. differentiation balance: Maize rbm48 mutants show smaller endosperm cells and failure of specialized cells like the basal endosperm transfer layer (BETL) to properly differentiate . This indicates RBM48 may regulate the balance between cell proliferation and differentiation in specific tissues.

• Gene expression changes: Differential gene expression analysis in maize rbm48 mutants identified 5771 differentially expressed transcripts, including nutrient reservoir genes and cell-type specific markers . These large-scale transcriptome changes reflect the substantial impact of U12 splicing on developmental gene expression programs.

• Developmental timing alterations: Expression analysis of developmental markers in maize rbm48 mutants showed both reduced expression and altered temporal patterns , suggesting RBM48 may be involved in the timing of developmental transitions.

For Pongo abelii specifically, researchers should investigate RBM48's role in primate-specific developmental processes, particularly in neural and germ cell differentiation where U12 introns are known to be enriched in other mammals.

What are the recommended expression systems for producing functional recombinant Pongo abelii RBM48?

For producing functional recombinant Pongo abelii RBM48, researchers should consider several expression systems with their respective advantages:

• Baculovirus expression system: This is often preferred for eukaryotic proteins requiring post-translational modifications. Commercial sources already use this system for related recombinant proteins . Benefits include high expression levels and proper protein folding.

• Mammalian expression systems: For studying proteins in a context closer to their native environment, mammalian cell lines (particularly primate cells) might better preserve species-specific post-translational modifications and folding patterns.

• Escherichia coli: While less ideal for proteins requiring complex modifications, bacterial expression can be optimized for RBM48 by:

  • Using strains enhanced for eukaryotic protein expression

  • Co-expressing chaperones to assist protein folding

  • Using fusion tags (His, GST, MBP) to improve solubility

  • Expressing separate domains independently if full-length expression is problematic

• Cell-free expression systems: These can be advantageous for producing proteins that might be toxic to host cells and allow immediate purification.

For any expression system, researchers should verify protein functionality through:

• RNA binding assays to confirm the RRM domain is properly folded

• Co-immunoprecipitation with known partners like ARMC7

• Splicing activity assays using in vitro splicing reactions with U12-containing pre-mRNAs

What are the optimal methods for studying RBM48-RNA interactions in vitro and in vivo?

For comprehensive analysis of RBM48-RNA interactions, researchers should consider complementary in vitro and in vivo approaches:

In vitro methods:

• RNA Electrophoretic Mobility Shift Assay (EMSA): Use purified recombinant Pongo abelii RBM48 and labeled RNA oligonucleotides containing predicted binding motifs to quantify binding affinity and specificity.

• Surface Plasmon Resonance (SPR): Determine binding kinetics and affinity constants of RBM48-RNA interactions in real-time.

• RNA Pull-down: Employ biotinylated RNA baits containing U12 intron sequences to capture RBM48 from cell extracts, followed by western blotting or mass spectrometry.

• Systematic Evolution of Ligands by Exponential Enrichment (SELEX): Identify the precise RNA sequence motifs recognized by RBM48's RRM domain.

In vivo methods:

• Cross-linking and Immunoprecipitation (CLIP): UV-crosslink RNA-protein complexes in living cells followed by immunoprecipitation with RBM48-specific antibodies and high-throughput sequencing to map binding sites genome-wide .

• Photoactivatable Ribonucleoside-Enhanced CLIP (PAR-CLIP): Incorporate photoreactive nucleoside analogs into cellular RNA to increase crosslinking efficiency and provide additional information about binding sites through characteristic mutation patterns.

• RNA Antisense Purification (RAP): Use biotinylated antisense oligos to capture specific RNA targets and identify associated proteins including RBM48.

• Proximity Labeling: Fuse RBM48 with enzymes like APEX2 or BioID to biotinylate nearby proteins and RNAs when activated, revealing the dynamic RBM48 interactome.

For all methods, appropriate controls including RBM48 mutants lacking the RRM domain should be included to validate binding specificity.

How can researchers quantify the impact of RBM48 on U12 intron splicing efficiency?

To quantify RBM48's impact on U12 intron splicing efficiency, researchers should implement the following analytical approaches:

• Percent Spliced Out (PSO) calculation: Calculate PSO values for U12 introns using the formula: PSO = (number of exon-exon junction reads) / (number of exon-exon junction reads + number of exon-intron junction reads) . This metric effectively quantifies splicing efficiency in control versus RBM48-depleted samples.

• Intron Retention Index (IRI): Measure the ratio of intronic reads to adjacent exonic reads, normalized to total read depth. An increased IRI after RBM48 depletion indicates reduced splicing efficiency.

• RT-PCR validation: Design primers spanning selected U12 introns and perform semi-quantitative or quantitative RT-PCR to validate RNA-seq findings, as done in maize rbm48 studies .

• High-resolution RNA-seq analysis: Use strand-specific, paired-end deep sequencing with sufficient coverage to detect rare splicing events. Apply specialized computational pipelines like MAJIQ or rMATS designed to detect differential splicing.

• Nascent RNA sequencing: Techniques like NET-seq or GRO-seq can capture RNA processing events as they occur, allowing temporal resolution of splicing defects.

• Statistical significance testing: Apply false discovery rate (FDR) correction for multiple testing when determining significantly affected introns. In maize studies, an FDR < 0.05 with a threshold of >20% ΔPSO identified U12 introns significantly affected by rbm48 mutations .

This multi-faceted approach would provide robust quantification of how Pongo abelii RBM48 affects U12 intron splicing across the transcriptome.

What approaches can identify the full spectrum of RBM48 protein interaction partners in Pongo abelii cells?

To comprehensively identify RBM48 protein interaction partners in Pongo abelii cells, researchers should employ multiple complementary approaches:

• Affinity Purification Mass Spectrometry (AP-MS): Express tagged RBM48 (FLAG, HA, or GFP) in Pongo abelii cells or closely related primate cells, perform immunoprecipitation under varying stringency conditions, and identify co-purifying proteins by mass spectrometry. This approach has successfully identified interactions between RBM48 and proteins like ARMC7 in other species .

• Proximity-dependent Biotin Identification (BioID): Fuse RBM48 to a biotin ligase (BirA*) that biotinylates nearby proteins, enabling identification of both stable and transient interactors in their native cellular environment.

• Cross-linking Mass Spectrometry (XL-MS): Use protein cross-linkers followed by mass spectrometry to capture and identify interacting proteins, providing additional information about interaction interfaces.

• Co-immunoprecipitation with targeted validation: Based on known interactions in other species (U2AF subunits, RGH3/ZRSR2, ARMC7) , perform targeted Co-IP experiments with antibodies against these specific candidates.

• Yeast Two-Hybrid screening: Use RBM48 as bait to screen Pongo abelii cDNA libraries, though this approach may miss interactions dependent on RNA or specific cellular contexts.

• Protein fragment complementation assays: Split reporter proteins (like luciferase or fluorescent proteins) are fused to potential interacting partners to validate interactions in living cells.

For all approaches, researchers should include appropriate controls (domain mutants, non-relevant proteins) to distinguish specific from non-specific interactions. Data from multiple methods should be integrated to build a high-confidence interactome network for Pongo abelii RBM48.

How can studies of Pongo abelii RBM48 contribute to understanding evolutionary conservation of RNA splicing mechanisms?

Studies of Pongo abelii RBM48 offer valuable opportunities to understand the evolutionary conservation of RNA splicing mechanisms, especially U12 splicing, for several key reasons:

• Evolutionary position: As a non-human great ape, Pongo abelii represents an important evolutionary branch point for comparing splicing mechanisms between humans and more distant primates. Comparative analyses of RBM48 sequence, structure, and function between orangutans, humans, and other primates can reveal evolutionary pressures on the U12 spliceosome.

• Conserved splicing machinery: The high conservation of RBM48 across species that maintain U12 introns suggests fundamental evolutionary constraints . Comparing binding specificities and protein interactions of Pongo abelii RBM48 with those in other species would reveal which aspects of U12 splicing are most evolutionary constrained.

• Species-specific adaptations: Any differences in RBM48 function between Pongo abelii and other species might reveal adaptive changes in splicing regulation. The co-selection patterns of RBM48 and RGH3/ZRSR2 observed across eukaryotes can be further refined by detailed analysis in closely related primates.

• Intron conservation patterns: By mapping which U12 introns are regulated by RBM48 in Pongo abelii compared to other species, researchers can identify evolutionarily conserved versus species-specific regulatory targets, providing insights into how minor splicing contributes to phenotypic diversity among primates.

• Protein interaction networks: Comparative analysis of RBM48 interaction partners across species, including the conserved interaction with ARMC7 , can reveal how splicing regulatory networks have evolved in primates.

These studies would contribute to our fundamental understanding of how essential cellular processes like RNA splicing balance evolutionary conservation with species-specific adaptations.

What are the implications of RBM48 research for understanding human splicing disorders?

Research on Pongo abelii RBM48 has significant implications for understanding human splicing disorders:

• Evolutionary models for disease: As our closest living relatives, great apes like Pongo abelii provide valuable evolutionary context for understanding human splicing disorders. Studies in orangutan RBM48 can help distinguish conserved disease-relevant mechanisms from human-specific aspects of splicing regulation.

• Minor spliceosome-related diseases: Several human disorders are associated with U12 splicing dysfunction, including myelodysplastic syndromes linked to mutations in ZRSR2 (RBM48's interaction partner) and microcephalic osteodysplastic primordial dwarfism type I (MOPD1) associated with U4atac snRNA mutations. Understanding RBM48's function across primates could provide new insights into these conditions.

• Conserved splicing mechanisms: Studies in maize show that RBM48 affects splicing of U12 introns in genes involved in diverse cellular processes . Identifying conserved RBM48-dependent U12 introns between Pongo abelii and humans could reveal critical splicing events relevant to human disease.

• Therapeutic development: Detailed understanding of RBM48's molecular function could inform the development of therapies for splicing disorders. For example, if RBM48 regulates specific U12 introns affected in human disease, it might represent a therapeutic target for modulating their splicing.

• Cell differentiation disorders: The role of RBM48 in promoting cell differentiation and repressing proliferation in maize suggests it may have similar functions in primates. Dysregulation of this balance is central to many human disorders, including developmental disorders and cancer.

By studying RBM48 in our evolutionary relatives, researchers can gain unique insights into the fundamental mechanisms underlying human splicing disorders that might not be apparent from human studies alone.

What quality control measures are essential when working with recombinant Pongo abelii RBM48?

When working with recombinant Pongo abelii RBM48, researchers should implement the following quality control measures to ensure experimental reliability:

• Protein purity assessment:

  • SDS-PAGE with Coomassie staining to verify size and purity (>95% purity recommended for functional studies)

  • Mass spectrometry to confirm protein identity and detect any post-translational modifications

  • Size exclusion chromatography to assess aggregation state and homogeneity

• Functional validation:

  • RNA binding assays to confirm that the RRM domain is properly folded and functional

  • Co-immunoprecipitation with known interaction partners like ARMC7 to verify the protein's ability to form expected complexes

  • Circular dichroism (CD) spectroscopy to assess secondary structure integrity

• Stability testing:

  • Thermal shift assays to determine protein stability under various buffer conditions

  • Time-course activity assays to assess functional stability during storage

  • Freeze-thaw testing to determine optimal aliquoting and storage protocols

• Contaminant testing:

  • Endotoxin testing, particularly important for protein produced in bacterial systems

  • Nuclease activity assays to ensure no contaminating nucleases are present

  • RNase-free validation for RNA interaction studies

• Batch consistency:

  • Lot-to-lot comparison using functional assays to ensure reproducibility

  • Reference standard inclusion in all analyses to track potential variability

Implementation of these quality control measures will ensure that experimental outcomes reflect the true biological properties of Pongo abelii RBM48 rather than artifacts of poor protein quality or contamination.

How should researchers address species-specific differences when extrapolating RBM48 findings between model organisms and Pongo abelii?

When extrapolating RBM48 findings between model organisms and Pongo abelii, researchers should systematically address species-specific differences using the following approaches:

• Sequence and structure comparison:

  • Perform detailed sequence alignments of RBM48 across species, focusing on functional domains (RRM, RS-rich region)

  • Use homology modeling to predict structural differences that might affect function

  • Identify species-specific post-translational modification sites that could alter activity

• Target intron analysis:

  • Compare U12 intron conservation across species

  • Determine whether orthologous genes have retained U12 introns in Pongo abelii versus model organisms

  • Analyze U12 splice site sequences for species-specific variations that might affect RBM48 binding

• Interactome comparisons:

  • Identify conserved versus species-specific protein interaction partners

  • Verify whether key interactions (like RBM48-ARMC7) are maintained in Pongo abelii

  • Perform comparative proteomics to identify primate-specific RBM48 interactors

• Functional validation:

  • Use cross-species complementation studies to determine functional equivalence

  • Test whether Pongo abelii RBM48 can rescue phenotypes in model organisms with RBM48 depletion

  • Perform side-by-side functional assays with RBM48 from different species

• Cellular context considerations:

  • Account for differences in cellular environments (e.g., primate-specific RNA binding proteins)

  • Consider tissue-specific expression patterns that might differ between species

  • Evaluate species-specific alternative splicing events that might affect RBM48 function

By systematically addressing these factors, researchers can make more reliable extrapolations while also identifying potentially important species-specific aspects of RBM48 biology in Pongo abelii.