Recombinant Rat Insulin-like growth factor 2 mRNA-binding protein 1 (Igf2bp1)

What is the domain structure of rat IGF2BP1 and how does it compare to human IGF2BP1?

Rat IGF2BP1, like its human counterpart, contains six RNA-binding domains: two N-terminal RNA recognition motif (RRM) domains (RRM1 and RRM2) and four C-terminal heterogeneous ribonucleoprotein K homology (KH) domains (KH1-4). The KH domains are arranged as pseudo-dimers (KH1-2 and KH3-4) connected via two intrinsically disordered linker regions . These disordered linkers contain important regulatory phosphorylation sites, including S181 in linker 1 and Y396 in linker 2, which modulate protein function. While human and rat IGF2BP1 share high sequence homology in their structured domains, species-specific differences may exist in the disordered regions, potentially affecting regulatory mechanisms.

How should I validate the complete knockout of IGF2BP1 in a rodent model?

Complete validation of IGF2BP1 knockout requires multiple approaches:

• Genomic verification: PCR confirmation of the targeting cassette insertion or deletion in the IGF2BP1 gene

• Transcript analysis: qPCR using primers directed at different regions (particularly the 5' region) to confirm reduction in IGF2BP1 mRNA

• Protein verification: Western blot analysis using antibodies against different epitopes (especially N-terminal) to ensure absence of full-length or truncated proteins

• Functional assays: Testing for known IGF2BP1-dependent phenotypes

For example, in one study, researchers confirmed IGF2BP1 knockout by PCR detection of a β-geo cassette inserted between exons 13-14, performed Western blot with multiple antibodies against the N-terminus, and conducted qPCR toward the 5' region to verify reduced mRNA quantities .

What phenotypes are observed in IGF2BP1 knockout mice?

IGF2BP1 knockout mice exhibit multiple phenotypes:

• Neonatal lethality: Complete knockout is often lethal, indicating essential developmental functions

• Neurological defects: Disorganization in the developing neocortex with loss of cortical marginal cell density at E17.5

• Migration defects: Fewer mitotically active cells in the cortical plate (measured via BrdU labeling)

• β-actin mRNA dysregulation: Significantly decreased endogenous β-actin mRNA transport and anchoring

• Cytoskeletal abnormalities: Increased actin protein content in neurons

These findings demonstrate that IGF2BP1 is essential for proper brain development and postnatal survival, with its absence leading to significant developmental abnormalities .

How should I design experiments to study IGF2BP1 target RNA interactions in rat tissues?

To study IGF2BP1-RNA interactions in rat tissues:

• Crosslinking and immunoprecipitation (CLIP) approaches:

  • CLIP-seq: UV crosslinking followed by immunoprecipitation and sequencing

  • iCLIP or PAR-CLIP: For higher resolution binding sites

  • eCLIP: Enhanced CLIP with controls for background

• RNA immunoprecipitation (RIP):

  • Use anti-IGF2BP1 antibodies with appropriate IgG controls

  • Confirm enrichment via immunoblotting of bead-bound fractions

  • Analyze ~2.5% of total lysate as input control and ~5% of flow-through

• Motif analysis:

  • Identify enriched motifs in bound RNA sequences

  • Compare with known IGF2BP1 binding motifs, typically 4-nucleotide recognition motifs for KH domains

• Validation experiments:

  • qPCR for candidate target RNAs

  • Luciferase reporter assays with wild-type and mutated binding sites

  • RNA stability assays with actinomycin D treatment

These methods can be applied to study tissue-specific IGF2BP1-RNA interactions, with appropriate controls to account for background binding .

What controls should be included when studying IGF2BP1 phosphorylation in rat cells?

When studying IGF2BP1 phosphorylation:

Additionally, include temporal controls to capture phosphorylation dynamics and subcellular fractionation to determine localization changes upon phosphorylation. For phosphorylation site mapping, combine targeted mass spectrometry with phospho-specific antibodies. Recent research has shown that phosphorylation at S181 and Y396 in the disordered linkers regulates IGF2BP1's ability to form ribonucleoprotein condensates, with differential effects on condensate size and dynamics .

How can I investigate IGF2BP1's role in ribonucleoprotein (RNP) condensate formation in vitro?

To investigate IGF2BP1's role in RNP condensate formation:

• Protein purification:

  • Express and purify recombinant rat IGF2BP1 (full-length and domain constructs)

  • Include wild-type and phospho-variant (S181E, Y396E) proteins

  • Ensure high purity (>90%) verified by SDS-PAGE and SEC

• In vitro phase separation assays:

  • Use fluorescently labeled proteins (e.g., mCherry-tagged IGF2BP1) at 5% of total protein

  • Include relevant RNA substrates (e.g., XBP1-derived 36 nt RNA)

  • Test different protein:RNA ratios and buffer conditions

  • Monitor condensate formation by fluorescence microscopy

• Quantitative analysis:

  • Measure condensate area, number, and intensity

  • Analyze condensate dynamics using FRAP (Fluorescence Recovery After Photobleaching)

  • Compare wild-type vs. phospho-variant behaviors

• Biophysical characterization:

  • Determine protein-RNA binding affinities using techniques like fluorescence anisotropy

  • Measure condensate material properties using techniques like microrheology

Recent research has shown that the S181E phosphomimetic mutant impairs condensate formation, while Y396E increases condensate size and affects dynamics, with median area per droplet of 10.8 μm and mean total area of 9298 μm² at 5 μM protein and RNA concentration for Y396E .

How can I distinguish between direct and indirect effects of IGF2BP1 on target mRNA stability?

To distinguish direct from indirect effects of IGF2BP1 on mRNA stability:

• Direct binding assessment:

  • CLIP-seq to identify direct binding sites on target mRNAs

  • Mutational analysis of predicted binding motifs

  • In vitro binding assays with purified components

• mRNA half-life measurements:

  • Actinomycin D chase experiments in wild-type vs. IGF2BP1-deficient cells

  • Pulse-chase labeling with 4-thiouridine (4sU) for nascent RNA

  • Compare decay rates of direct targets vs. non-targets

• Mechanism dissection:

  • Tethering assays: Fuse IGF2BP1 to MS2 coat protein and express reporter mRNAs with MS2 binding sites

  • Domain deletion/mutation studies to identify regions required for stability effects

  • miRNA dependency: Test if effects require miRNA pathway components (e.g., AGO2)

• Reconstitution experiments:

  • Add back wild-type or mutant IGF2BP1 to knockout cells

  • Test if stability is restored for putative direct targets

  • Use inducible systems for temporal control

For example, research has shown that IGF2BP1 stabilizes E2F1 mRNA by binding to its 3'UTR and interfering with miR-93-5p-mediated regulation, as demonstrated by reduced luciferase reporter activity and increased AGO2-E2F1 mRNA association in IGF2BP1-knockout cells .

How should I analyze and interpret RNA-seq data from IGF2BP1-deficient rat models?

When analyzing RNA-seq data from IGF2BP1-deficient models:

• Differential expression analysis:

  • Compare wild-type vs. IGF2BP1-deficient samples

  • Use appropriate statistical methods (DESeq2, edgeR, limma)

  • Apply multiple testing correction (FDR < 0.05)

  • Consider fold-change thresholds (|log2FC| > 1.5 or 2.0)

• Target enrichment analysis:

  • Cross-reference with IGF2BP1 CLIP-seq data

  • Compare changes in direct targets vs. non-targets

  • Analyze mRNA features (3'UTR length, miRNA sites, m6A sites)

• Pathway and functional analysis:

  • Perform GO term and KEGG pathway enrichment

  • Use GSEA for detecting coordinated changes

  • Consider Reactome pathway analysis

• Integration with other data types:

  • Compare with proteomics data to identify translational effects

  • Correlate with m6A-seq data since IGF2BP1 is an m6A reader

In published studies, IGF2BP1 depletion affected thousands of genes, with 2,405 differentially expressed genes identified in one study, including 2,199 upregulated and 206 downregulated genes. When filtering with |log2FC| > 1.5, 875 genes were identified (828 upregulated, 47 downregulated) .

What statistical approaches are most appropriate for analyzing IGF2BP1-RNA interactions from CLIP-seq experiments?

For statistical analysis of IGF2BP1 CLIP-seq data:

• Peak calling and normalization:

  • Use specialized tools (PARalyzer, CLIPper, Piranha)

  • Account for input/background signal and crosslinking biases

  • Consider biological replicates for robust peak identification

• Motif discovery:

  • Apply de novo motif finding algorithms (MEME, HOMER)

  • Compare enriched motifs with known IGF2BP1 binding preferences

  • Analyze positional distribution of motifs relative to peaks

• Differential binding analysis:

  • Compare binding across conditions using DESeq2 or edgeR

  • Calculate fold-enrichment over background

  • Apply appropriate multiple testing correction

• Integration with genomic features:

  • Analyze distribution across transcript regions (5'UTR, CDS, 3'UTR)

  • Correlate with RNA modifications (especially m6A)

  • Assess proximity to miRNA binding sites

• Correlation with expression changes:

  • Calculate Pearson or Spearman correlations between binding and expression changes

  • Perform regression analysis to identify predictive features

For comprehensive analysis, combine data from multiple CLIP protocols and cell types. Previous studies analyzed IGF2BP1 binding using multiple datasets: two PAR-CLIP (HEK293), two eCLIP (hESCs), two eCLIP (HepG2), and two eCLIP (K562) datasets to identify robust binding sites across different cellular contexts .

How can IGF2BP1 knockout rat models inform our understanding of intestinal disorders?

IGF2BP1 knockout rat models provide valuable insights into intestinal disorders:

• Inflammatory conditions:

  • IGF2BP1 deletion in intestinal epithelial cells causes mild active colitis and mild-to-moderate active enteritis

  • Knockout aggravates dextran sodium sulfate-induced colitis

  • These phenotypes model aspects of inflammatory bowel disease

• Barrier function:

  • IGF2BP1 removal compromises intestinal epithelial barrier function

  • This results from altered protein expression at tight junctions

  • Specifically, IGF2BP1 stabilizes occludin (Ocln) mRNA, a key tight junction protein

• Intervention testing:

  • IGF2BP1 knockout models allow testing of therapeutic approaches

  • Ectopic occludin expression in IGF2BP1-knockdown cells restores barrier function

  • This suggests occludin as a potential therapeutic target

• Experimental approaches:

  • Use Villin-CreERT2:Igf2bp1flox/flox mice for inducible, intestine-specific knockout

  • Measure gut barrier and epithelial permeability

  • Employ biochemical approaches to identify direct targets

These findings establish IGF2BP1-dependent regulation of occludin expression as an important mechanism in intestinal barrier function maintenance and prevention of colitis .

What methods should I use to study IGF2BP1's roles in rat cancer models?

To study IGF2BP1 in rat cancer models:

• Expression analysis in tumors:

  • Immunohistochemistry for protein localization

  • qRT-PCR for transcript levels

  • Western blotting for protein expression

  • Compare with matched normal tissues

• Functional investigations:

  • Generate IGF2BP1-overexpressing and knockout/knockdown rat cell lines

  • Analyze:

    • Proliferation (doubling time, colony formation)

    • Cell cycle progression (flow cytometry)

    • Migration and invasion (transwell assays)

    • Clonogenicity

• Molecular mechanism studies:

  • Identify cancer-relevant target mRNAs (RIP-seq, CLIP-seq)

  • Analyze pathway enrichment (E2F-driven genes, Wnt/β-catenin targets)

  • Investigate post-transcriptional effects using luciferase reporters

  • Study m6A dependency using m6A-RIP-seq

• In vivo approaches:

  • Establish xenograft models with IGF2BP1-modified cells

  • Use inducible systems for temporal control

  • Test small molecule inhibitors (e.g., BTYNB)

  • Analyze tumor growth, invasion, and metastasis

Research has shown that IGF2BP1 acts as a post-transcriptional super-enhancer of E2F-driven gene expression in cancer, promoting G1/S cell cycle transition by stabilizing mRNAs encoding positive regulators like E2F1. The small molecule BTYNB disrupts this function by impairing IGF2BP1-RNA association, interfering with E2F-driven gene expression and tumor growth in mouse models .

How do the functions of recombinant rat IGF2BP1 compare to IGF2BP2 and IGF2BP3 in similar experimental systems?

Comparison of IGF2BP family members in experimental systems:

The IGF2BP family members show both redundant and non-redundant functions. For example, in HIV-1 experiments, IGF2BP1 showed moderate inhibition of virus production, IGF2BP2 demonstrated stronger inhibition, while IGF2BP3 had no effect on viral infectivity . This suggests distinct roles despite structural similarities. When designing experiments using recombinant rat IGF2BP1, consider potential compensatory effects from other family members, which may influence interpretation of knockdown/knockout phenotypes.

How does phosphorylation of rat IGF2BP1 affect its function compared to other post-translational modifications?

Comparison of IGF2BP1 post-translational modifications:

Phosphorylation and citrullination have been more extensively studied than other modifications. Phosphorylation at S181 in linker 1 impairs condensate formation while Y396E in linker 2 increases condensate size and dynamics . Citrullination at R167 promotes rheumatoid synovial aggression by enhancing SEMA3D mRNA stability through improved interaction with ELAVL1 . These distinct modifications provide multiple regulatory layers for IGF2BP1 function, creating context-specific effects on RNA metabolism.

What are the key technical challenges in producing functional recombinant rat IGF2BP1 protein?

Key challenges in producing functional recombinant rat IGF2BP1:

• Expression system selection:

  • E. coli: Simple but lacks post-translational modifications

  • Insect cells: Better folding but moderate yield

  • Mammalian cells (HEK293): Proper modifications but expensive

  • Cell-free systems: Rapid but potential folding issues

• Solubility and purification:

  • Full-length protein (577 amino acids) prone to aggregation

  • Multiple RNA-binding domains complicate folding

  • Requires optimization of buffer conditions (typically higher salt)

  • Consider fusion tags (His, GST, Strep) for purification

• RNA contamination:

  • High affinity for RNA leads to co-purification of bacterial/host RNAs

  • RNase treatment may be necessary but can affect protein structure

  • High salt washes (500-750 mM NaCl) can reduce RNA binding

• Maintaining functionality:

  • Verifying proper folding using circular dichroism

  • RNA-binding assays to confirm activity

  • Testing for condensate formation capability

  • Phosphorylation status affects function

• Storage and stability:

  • Prone to aggregation during freeze-thaw cycles

  • Consider flash-freezing aliquots in liquid nitrogen

  • Add glycerol (10-15%) to storage buffer

  • Test for activity after storage

For optimal results, expression in HEK-293 cells with His-tag purification can achieve >90% purity as determined by Bis-Tris PAGE, anti-tag ELISA, Western Blot, and analytical SEC .

How can I overcome challenges in studying IGF2BP1-mediated effects in complex rat tissue systems?

Strategies to overcome challenges in studying IGF2BP1 in complex tissues:

• Cell-type heterogeneity:

  • Use single-cell techniques (scRNA-seq) to resolve cell-type specific effects

  • Employ cell-type specific markers for co-localization studies

  • Consider cell sorting (FACS) before molecular analyses

  • Use conditional knockout models with cell-type specific Cre drivers

• Distinguishing direct from indirect effects:

  • Combine CLIP-seq with RNA-seq from the same tissues

  • Use acute depletion systems (e.g., auxin-inducible degron) to capture primary effects

  • Apply network analysis to identify direct vs. downstream effects

  • Consider ex vivo culture systems for controlled manipulations

• Functional redundancy with other IGF2BPs:

  • Design experiments to account for compensatory mechanisms

  • Consider double/triple knockouts where feasible

  • Use domain-specific approaches to target unique functions

  • Employ rescue experiments with paralogs to test specificity

• Technical approaches:

  • For tissue sections: RNAscope combined with immunofluorescence

  • For biochemical studies: Optimize tissue-specific extraction protocols

  • For in vivo monitoring: Consider CRISPR-based endogenous tagging

  • For target validation: Use tissue-specific AAV delivery of shRNAs

When analyzing single-cell data, be aware of potential biases from enzymatic digestion and capture. In previous studies, researchers noted that "accurately determining the proportions of different germ cell types using single-cell data may be challenging" .

What emerging technologies will advance our understanding of rat IGF2BP1 function in development and disease?

Emerging technologies for IGF2BP1 research:

• Spatial transcriptomics:

  • Visium or Slide-seq to map IGF2BP1 targets in tissue context

  • Spatial CITE-seq for simultaneous protein and RNA profiling

  • MERFISH for subcellular RNA localization patterns

• Live-cell RNA imaging:

  • MS2/PP7 systems to track IGF2BP1-bound mRNAs in real-time

  • CRISPR-Cas13 RNA tracking without genetic modification of targets

  • Optogenetic control of IGF2BP1 activity for temporal precision

• Structural biology advances:

  • Cryo-EM to visualize IGF2BP1-RNA complexes

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) for dynamics

  • AlphaFold2/RoseTTAFold predictions integrated with experimental data

• High-throughput functional screens:

  • CRISPR activation/repression screens for IGF2BP1 regulatory networks

  • RNA-binding domain mutagenesis with deep mutational scanning

  • Synthetic RNA libraries to define binding preferences

• Organoid and advanced in vitro models:

  • Brain organoids to study neurodevelopmental functions

  • Intestinal organoids to investigate barrier functions

  • Microfluidic systems for spatial RNA localization studies

These technologies will provide unprecedented insights into IGF2BP1's roles in normal development and disease states, particularly in previously challenging contexts like the dynamic regulation of RNP granules and subcellular RNA localization.

How might targeting IGF2BP1 in rats inform therapeutic approaches for human diseases?

Therapeutic implications from rat IGF2BP1 research:

• Cancer therapeutics:

  • Small molecule inhibitors: Building on BTYNB to develop rat-specific and human-specific compounds

  • Targeting condensate formation: Compounds that disrupt phase separation

  • Degraders: PROTAC approaches for selective IGF2BP1 degradation

  • miRNA-based therapies: Enhancing natural negative regulators

• Inflammatory diseases:

  • Intestinal barrier restoration: Targeting IGF2BP1-occludin axis

  • Rheumatoid arthritis: Inhibiting citrullination of IGF2BP1

  • Modulating immune response: IGF2BP1-dependent inflammatory pathways

• Neurodevelopmental disorders:

  • RNA localization modulators: Compounds affecting IGF2BP1-mediated mRNA transport

  • Axon guidance: Therapies targeting IGF2BP1's role in neuronal migration

  • Synaptic plasticity: Approaches to modulate activity-dependent localization

• Translational considerations:

  • Comparative studies between rat and human IGF2BP1

  • Humanized rat models expressing human IGF2BP1

  • Identification of conserved vs. species-specific mechanisms

• Biomarkers:

  • Post-translational modifications of IGF2BP1 as disease indicators

  • IGF2BP1 target RNA profiles as diagnostic tools

  • Circulating IGF2BP1 autoantibodies in autoimmune conditions