RANBP1 Human

What is the primary cellular function of RANBP1 in humans?

RANBP1 functions primarily as a regulator of the Ran GTPase pathway, which controls nucleocytoplasmic transport and mitotic spindle assembly. It specifically interacts with GTP-bound Ran (Ran-GTP) but not with GDP-bound Ran (Ran-GDP), acting as a co-activator of Ran GTPase activity . RANBP1 works in conjunction with RANGAP1 (Ran GTPase-Activating Protein 1) to increase GTP hydrolysis, thereby regulating the cycling between Ran-GTP and Ran-GDP states .

RANBP1 plays essential roles in maintaining the nucleus-cytoplasmic gradient, which is crucial for proper nuclear import and export of proteins, nucleic acids, and microRNAs . It controls mitotic spindle assembly and ensures accurate chromosome segregation during cell division . Additionally, RANBP1 regulates the interaction between Ran and transport receptors such as importin beta and exportin 1 (XPO1), alleviates TNPO1-dependent inhibition of RAN GTPase activity, and promotes the dissociation of various protein complexes involved in nuclear transport .

These diverse functions collectively contribute to cellular homeostasis, proper cell division, and epigenomic regulation, positioning RANBP1 as a crucial player in fundamental cellular processes.

How does RANBP1 regulate the Ran GTPase pathway?

RANBP1 regulates the Ran GTPase pathway through several well-characterized molecular mechanisms:

• Co-activation of GTP Hydrolysis: RANBP1 does not increase RAN GTPase activity by itself but significantly enhances GTP hydrolysis mediated by RANGAP1 . This co-activator function is critical for maintaining the proper cycling between Ran-GTP and Ran-GDP states.

• Inhibition of Nucleotide Exchange: RANBP1 inhibits RCC1 (Regulator of Chromatin Condensation 1)-dependent exchange of Ran-bound GDP by GTP . It complexes with Ran-GTP and RCC1-Ran, holding the latter complex in a less responsive state for guanine nucleotide exchange on Ran .

• Complex Disassembly: RANBP1 promotes the disassembly of complexes formed by Ran and importin beta, Ran and KPNA2/CSE1L, and induces conformational changes in the XPO1-Ran complex that trigger the release of cargo proteins' nuclear export signals .

• Balance Maintenance: The ratio of RANBP1 to RCC1 has been identified as a pivotal point for normal cell cycle progression. Experimental evidence shows that altering this protein ratio leads to defects in DNA replication, nuclear assembly, and nuclear transport of proteins .

What is the tissue distribution of RANBP1 in the human body?

RANBP1 displays a broad but differential tissue distribution pattern across human tissues:

This wide but differential tissue distribution pattern suggests tissue-specific functions of RANBP1 beyond its core roles in nucleocytoplasmic transport and mitotic regulation, potentially contributing to specialized cellular functions in different tissues.

What experimental methods are commonly used to study RANBP1 expression?

Researchers employ various experimental methods to study RANBP1 expression and function:

• Transcriptomic Analysis:

  • RNA sequencing (RNA-seq) to quantify RANBP1 mRNA expression levels

  • Analysis of transcriptomic data from databases such as TCGA and GTEx

• Protein Detection Methods:

  • Western blotting (WB) using specific antibodies against RANBP1

  • Immunocytochemistry and immunofluorescence (ICC/IF) to visualize cellular localization

  • Mass spectrometry for detection in complex biological samples

• Functional Assays:

  • Fluorescence recovery after photobleaching (FRAP) and Fluorescence loss in photobleaching (FLIP) to study RANBP1 dynamics

  • Auxin-induced degron (AID) systems for targeted protein degradation studies

• Expression Databases and Resources:

  • HCCDB (Integrative Molecular Database for Hepatocellular Carcinoma) for expression patterns in HCC

  • Human Protein Atlas (HPA) for protein expression across tissues using immunohistochemistry

• Methylation Analysis:

  • MethSurv database to evaluate the prognostic value of RANBP1 methylation

These diverse methodological approaches allow researchers to comprehensively characterize RANBP1 expression patterns, localization, and functional implications in various biological contexts.

How does RANBP1 contribute to cell cycle regulation?

RANBP1 contributes to cell cycle regulation through multiple mechanisms:

• Mitotic Spindle Assembly:

  • RANBP1 controls mitotic RCC1 dynamics in human somatic tissue culture cells

  • It ensures proper localization of spindle assembly factors (SAFs), such as Hepatoma Up-Regulated Protein (HURP)

  • RANBP1 is required for normal mitotic spindle assembly and progression through mitosis via its effect on RAN

• Chromatin-RAN Gradient Regulation:

  • During mitosis, RANBP1 helps maintain the gradient of Ran-GTP (concentrated near mitotic chromatin) and Ran-GDP (more abundant distal to chromosomes)

  • This gradient spatially controls spindle formation by locally releasing SAFs from inhibitory interactions near chromosomes

• Cell Cycle Checkpoint Control:

  • RANBP1 plays a crucial role in spindle checkpoint formation and nucleation

  • Gene set enrichment analysis has shown enrichment of G2M-checkpoint, Wnt cell signaling, and DNA repair in RANBP1 high-expression phenotypes

• Regulation of RCC1-RAN Interactions:

  • The balance of the RANBP1/RCC1 ratio is pivotal for normal cell cycle progression

  • Altering this protein ratio leads to defects in DNA replication and nuclear assembly

Through these mechanisms, RANBP1 coordinates nuclear envelope breakdown, spindle formation, and chromosomal segregation during mitosis, ensuring genomic stability and proper cell division.

What are the challenges in studying the RanBP1-RCC1-Ran interaction dynamics in living cells?

Studying the RanBP1-RCC1-Ran interaction dynamics in living cells presents several methodological and conceptual challenges:

• Spatiotemporal Resolution Limitations:

  • RanBP1-RCC1-Ran interactions occur rapidly and dynamically, requiring high-resolution imaging techniques

  • Traditional fluorescence microscopy may lack the temporal resolution to track fast exchange rates

• Complex Formation Visualization:

  • The RRR complex (RCC1/Ran/RanBP1 heterotrimeric complex) forms and dissociates rapidly

  • Current methods may not adequately distinguish between different complex states

• Distinguishing Direct and Indirect Effects:

  • When manipulating RanBP1 levels, it's difficult to determine whether observed phenotypes are due to direct effects on RCC1-Ran interactions or indirect effects

  • Experimental depletion of RanBP1 in Xenopus egg extract samples resulted in a decrease in RCC1 as well, complicating interpretation

• Maintaining Physiological Relevance:

  • Overexpression or depletion approaches may disrupt the critical RANBP1/RCC1 ratio

  • The balance between these proteins is more important than their absolute levels

• Technical Approach Limitations:

  • Fluorescence-based techniques like FRAP and FLIP can measure protein mobility but may not directly reveal molecular interactions

  • Protein tagging for visualization may interfere with protein function

• Cell Cycle Dependency:

  • The dynamics and importance of these interactions vary throughout the cell cycle

  • Interactions during mitosis versus interphase may have different characteristics

Researchers are addressing these challenges through advanced methodologies including FRET-based biosensors, optogenetic approaches, super-resolution microscopy, and computational modeling to better understand these complex interactions.

How does RANBP1 overexpression impact mitotic spindle formation and chromosomal stability?

RANBP1 overexpression significantly impacts mitotic spindle formation and chromosomal stability through several interconnected mechanisms:

• Disruption of the Ran-GTP Gradient:

  • RANBP1 overexpression alters the spatial distribution of Ran-GTP around chromosomes during mitosis

  • This disruption affects the localized release of spindle assembly factors (SAFs)

• Altered RCC1 Dynamics on Chromatin:

  • Research has shown that RANBP1 controls mitotic RCC1 dynamics in human somatic tissue culture cells

  • Overexpression changes the association/dissociation rates of RCC1 with chromatin, affecting local Ran-GTP production

• Mislocalization of Spindle Assembly Factors:

  • Studies have observed the re-localization of HURP in metaphase cells after RanBP1 manipulation

  • This indicates that altered RCC1 dynamics functionally modulate SAF activities, potentially leading to defective kinetochore-microtubule attachments

• Cell Cycle Checkpoint Disruption:

  • RANBP1 overexpression is associated with G2M-checkpoint pathways and DNA repair mechanisms

  • Disruption of these checkpoints can allow cells with spindle defects to proceed through mitosis

• Chromosomal Instability Consequences:

  • The combined effects of altered spindle formation and checkpoint dysfunction can lead to:

    • Chromosome missegregation

    • Aneuploidy

    • Micronuclei formation

    • Genomic instability

• Impact on Mitotic Timing:

  • Proper RANBP1 function is required for normal progression through mitosis

  • Overexpression may disrupt the coordinated sequence of events necessary for accurate chromosome segregation

These mechanisms explain how RANBP1 overexpression, frequently observed in cancer cells, contributes to chromosomal instability and potentially to cancer progression.

What mechanisms explain the association between RANBP1 expression and poor prognosis in hepatocellular carcinoma?

The association between RANBP1 overexpression and poor prognosis in hepatocellular carcinoma (HCC) can be explained through several interconnected molecular mechanisms:

Understanding these mechanisms provides insights into potential therapeutic strategies targeting RANBP1 or its downstream effectors in HCC.

How can researchers effectively distinguish between the direct and indirect effects of RANBP1 in nucleocytoplasmic transport studies?

Distinguishing between direct and indirect effects of RANBP1 in nucleocytoplasmic transport studies requires sophisticated methodological approaches:

• Structure-Function Relationship Analysis:

  • Develop and utilize RANBP1 mutants that selectively disrupt specific protein-protein interactions

  • Compare phenotypes between wild-type and mutant proteins to isolate direct effects

• Temporal Resolution Approaches:

  • Employ rapid protein degradation systems like the Auxin-induced degron (AID) system

  • Monitor immediate changes (likely direct effects) versus delayed changes (potentially indirect effects)

• Spatial Manipulation Techniques:

  • Use optogenetic tools to activate or inhibit RANBP1 function with high spatial precision

  • Determine if effects are localized to the manipulation site (direct) or observed throughout the cell (possibly indirect)

• Biochemical Reconstitution:

  • Utilize in vitro transport assays with purified components to establish direct biochemical requirements

  • Compare results from minimal reconstituted systems to more complex cellular environments

• Quantitative Binding Studies:

  • Employ techniques like isothermal titration calorimetry or microscale thermophoresis to measure binding affinities

  • Correlate binding parameters with functional outcomes in transport assays

• Live-Cell Imaging with Multi-Color Labeling:

  • Simultaneously track RANBP1, Ran, transport receptors, and cargo proteins

  • Analyze the temporal sequence of interactions to establish causality

• Computational Modeling:

  • Develop mathematical models of the nucleocytoplasmic transport system

  • Simulate the effects of RANBP1 perturbations and compare with experimental data

• Correlation vs. Causation Analysis:

  • Distinguish between proteins whose localization merely correlates with RANBP1 manipulation versus those directly affected

  • This is particularly important when studying the RCC1-RAN-RANBP1 relationship

By combining these approaches, researchers can more confidently attribute specific nucleocytoplasmic transport phenotypes to direct RANBP1 functions versus downstream consequences.

What are the current approaches for targeting RANBP1 in cancer research?

Based on our understanding of RANBP1's roles in cellular processes and its association with cancer, several approaches for targeting RANBP1 in cancer research have emerged:

• Gene Expression Modulation:

  • RNA interference techniques to knockdown RANBP1 expression in cancer cells

  • CRISPR-Cas9 genome editing to create RANBP1 knockout models

  • These approaches help validate RANBP1 as a potential therapeutic target

• Protein-Protein Interaction (PPI) Inhibitors:

  • Development of small molecules that specifically disrupt the interaction between RANBP1 and RAN-GTP

  • Focus on compounds that prevent RANBP1 from enhancing RANGAP1-mediated GTP hydrolysis

• Combination Therapy Approaches:

  • Exploring synergistic effects between RANBP1 inhibition and conventional chemotherapeutics, cell cycle checkpoint inhibitors, or DNA damage response modulators

  • This is supported by GSEA showing enrichment of G2M-checkpoint and DNA repair pathways in RANBP1 high-expression phenotypes

• Biomarker Development:

  • Using RANBP1 expression and methylation status as predictive biomarkers for treatment response

  • Stratifying patients based on RANBP1 levels to identify those most likely to benefit from specific therapeutic approaches

• Immunomodulatory Strategies:

  • Targeting the altered immune microenvironment associated with RANBP1 overexpression

  • Developing approaches to counteract the immunomodulatory effects, particularly the influence on T helper cell populations

• Epigenetic Modulation:

  • Therapeutic strategies targeting the epigenetic regulation of RANBP1, given that its methylation status correlates with survival outcomes

These diverse approaches reflect the complex roles of RANBP1 in cancer biology and offer multiple potential avenues for therapeutic intervention.

How does methylation status of RANBP1 correlate with its expression and function in different cellular contexts?

The methylation status of RANBP1 represents an important epigenetic regulatory mechanism with significant clinical implications:

Understanding these complex relationships between RANBP1 methylation, expression, and function could reveal new opportunities for diagnostic and therapeutic strategies in cancer and other diseases.

What are the methodological considerations when analyzing RANBP1's role in immune cell infiltration?

Analyzing RANBP1's role in immune cell infiltration requires careful methodological considerations:

• Appropriate Immune Cell Deconvolution Methods:

  • Single-sample Gene Set Enrichment Analysis (ssGSEA) has been used for immune cell infiltration analysis

  • Researchers should consider multiple computational deconvolution methods to increase confidence in results

• Selection of Immune Cell Markers:

  • Using updated and well-validated marker gene sets is crucial for accurate immune cell type identification

  • The referenced study used marker genes for 24 immune cells derived from Bindea et al.

• Correlation vs. Causation Analysis:

  • Research identified correlations between RANBP1 expression and specific immune cell types (positive correlation with Th1, negative with Th17)

  • Experimental validation is necessary to establish whether RANBP1 directly influences immune cell recruitment

• Spatial Context Considerations:

  • Bulk tissue analysis loses spatial information about immune cell distribution

  • Technologies like spatial transcriptomics or multiplex immunohistochemistry can provide insights into spatial relationships

• Direct vs. Indirect Effects on Immune Function:

  • RANBP1's known functions in nucleocytoplasmic transport may indirectly affect immune signaling

  • Researchers should design experiments that can distinguish between direct effects on immune cells versus indirect effects

• Statistical Analysis Approaches:

  • Wilcoxon rank sum test and Spearman's correlation test have been employed to analyze immune cell associations

  • Multiple testing correction is essential when examining correlations with numerous immune cell types

• Integrated Multi-omics Analysis:

  • Combining RNA expression data with protein-level validation and functional assays provides more robust insights

  • Consider analyzing RANBP1's relationship with immune checkpoint molecules, cytokines, and chemokines

By addressing these methodological considerations, researchers can more rigorously assess RANBP1's role in shaping the tumor immune microenvironment and potentially identify new therapeutic strategies.

How can researchers differentiate between RANBP1-dependent and RANBP1-independent functions of RAN?

Differentiating between RANBP1-dependent and RANBP1-independent functions of RAN requires sophisticated experimental approaches:

• Domain-Specific Mutational Analysis:

  • Generate RAN mutants that specifically disrupt interaction with RANBP1 while preserving other functions

  • Experimental evidence supports the existence of separate domains in the RAN protein: one dependent on RANBP1 (essential for cell cycle progression and RNA export) and another independent from RANBP1 (influences nuclear protein import)

• Conditional RANBP1 Depletion Systems:

  • Utilize rapid and controllable protein degradation systems like the Auxin-induced degron (AID) system

  • Monitor which RAN functions are immediately affected versus those that persist after RANBP1 depletion

• Biochemical Reconstitution Assays:

  • Perform in vitro assays with purified components to test RAN functions in the presence and absence of RANBP1

  • Systematically add other factors to identify which combinations are necessary for specific functions

• Comparative Analysis Across Species:

  • Exploit natural variations in RANBP1-RAN interactions across evolutionary distant species

  • Compare findings from mammalian cells with those from Xenopus egg extracts

• Spatiotemporal Analysis:

  • Since RANBP1 is primarily cytoplasmic, functions of RAN in nuclear compartments may be less directly dependent on RANBP1

  • Compartment-specific analyses can help map the spatial boundaries of RANBP1 dependency

• Pathway-Specific Functional Assays:

  • Develop assays that specifically measure distinct RAN functions:

    • Nuclear import assays

    • Nuclear export assays

    • Mitotic spindle assembly assays

    • Nuclear envelope reformation assays

• Cell Cycle Analysis:

  • Given that RANBP1-dependent domains are essential for cell cycle progression, carefully designed synchronization experiments can help separate cell cycle-specific functions

By systematically employing these approaches, researchers can build a comprehensive map of RAN functions that explicitly identifies which processes require RANBP1 and which operate through alternative mechanisms.