Recombinant Xenopus laevis Protein RMD5 homolog A (rmnd5a)

What is the evolutionary conservation pattern of RMND5A across species?

RMND5A demonstrates significant conservation throughout the eukaryotic kingdom. Phylogenetic analysis reveals that Gid2/RMND5 proteins form distinct evolutionary branches. In vertebrates, two isoforms exist: RMND5A and RMND5B. The Xenopus laevis Rmnd5 protein is more closely related to mammalian RMND5A, showing 94% identity with human RMND5A compared to only 70% identity with human RMND5B . This high degree of conservation suggests evolutionary pressure to maintain RMND5A function, indicating its fundamental biological importance in cellular processes across species.

How does RMND5A function as an E3 ubiquitin ligase?

RMND5A contains several characteristic domains including the Lis homology (LisH), C-terminal to LisH, CT11-RanBPM, and E3 typical RING domains that enable its ubiquitin ligase function . As an E3 ubiquitin ligase, RMND5A combines with E1 and E2 enzymes to catalyze protein ubiquitination, marking specific target proteins for proteasomal degradation . In Xenopus laevis, Rmnd5 functions within the context of the CTLH complex, targeting unknown factors for polyubiquitination and subsequent degradation . This targeted protein degradation mechanism appears to be particularly important for proper brain development and potentially for regulating cell migration in various contexts.

What is the subcellular localization pattern of RMND5A?

Studies have demonstrated that human RMND5A localizes to both the cytosol and nucleus, whereas human RMND5B (the paralog) is entirely cytosolic . This differential localization suggests distinct functions for these paralogous proteins despite their structural similarities. The dual localization of RMND5A to both cytoplasmic and nuclear compartments indicates that it may have roles in regulating proteins in multiple cellular compartments, potentially affecting both cytoplasmic signaling pathways and nuclear processes such as transcription or chromatin organization.

What methods are effective for overexpressing RMND5A in experimental models?

For effective RMND5A overexpression, researchers have successfully employed lentiviral vector systems. A methodology from recent studies involves constructing overexpression plasmids using the pHS-AVC (pLV-hef1a-mScarlet-P2A-neo-WPRE-CMV) vector based on the human RMND5A gene sequence (NM_022780.4) . For viral particle packaging, HEK293 cells cultured in 6-cm dishes can be transfected with a mixture of recombinant plasmids, PAX2, and VSVG using FuGENE® 6 transfection reagent. After a 48-hour incubation period, lentiviral particles can be collected from the culture media. Target cells can then be infected with these particles in the presence of 10 μg/ml polybrene for 4 hours, followed by verification of overexpression using RT-qPCR and western blot analyses after an additional 48-hour incubation .

How can researchers effectively quantify RMND5A expression levels?

Accurate quantification of RMND5A expression can be achieved through RT-qPCR using specific primers. Based on published research, the following primers have been validated for RMND5A detection:

• Forward primer: 5′-AATGAGGTGATGGTGGAGCA-3′

• Reverse primer: 5′-GCATTTCCCGGTTTGACACT-3′

For normalization, GAPDH can be used as a reference gene with the following primers:

• Forward primer: 5′-GGAGCGAGATCCCTCCAAAAT-3′

• Reverse primer: 5′-GGCTGTTGTCATACTTCTCATGG-3′

Additional validation using western blot analysis with specific antibodies against RMND5A provides protein-level confirmation. This multi-level approach to quantification ensures reliable assessment of both transcript and protein expression levels.

What are the methodological considerations for studying RMND5A in Xenopus laevis models?

When working with Xenopus laevis models to study RMND5A, researchers should focus on stages relevant to neuronal development since rmnd5 expression is strongest in neuronal ectoderm, prospective brain, eyes, and ciliated cells of the skin . Careful staging of embryos according to standard developmental tables is critical. For loss-of-function studies, morpholino oligonucleotides targeting rmnd5 have been successfully used to suppress its function, resulting in observable malformations of the fore- and midbrain that can be assessed through morphological analysis . Gene expression analysis should include neuronal markers to assess the impact of RMND5A modulation on neural development. Additionally, researchers should consider the high molecular weight complex in which Rmnd5 is embedded when designing biochemical experiments to characterize its function in this model organism.

What is the functional relationship between miR-590-5p and RMND5A in cancer cells?

miR-590-5p has been identified as a microRNA that directly targets RMND5A. Comprehensive in silico analysis using multiple databases (TargetScan, miRDB, miRcode, and TarBase) identified miR-590-5p among six potential miRNAs that could target RMND5A . The 3′ untranslated region (UTR) of both miR-590-5p and RMND5A are highly conserved among mammals, and the mature miR-590-5p seeding region exhibits perfect alignment with the RMND5A 3′UTR .

Experimental validation showed that overexpression of miR-590-5p in pancreatic cancer cell lines (AsPC-1 and PANC-1) significantly decreased RMND5A expression at both mRNA and protein levels . Functionally, miR-590-5p mimics decreased migration ability in these cell lines and attenuated the promoting effects of RMND5A overexpression on cell migration . This regulatory relationship represents a potential therapeutic target, as modulating miR-590-5p levels could potentially control RMND5A expression and its downstream effects on cancer cell migration.

What methodologies are most effective for investigating RMND5A's role in cancer cell migration?

The wound healing assay has proven effective for investigating RMND5A's influence on cancer cell migration. In this approach, cancer cells (such as AsPC-1 and PANC-1 lines) are cultured to confluence, a "wound" is created by scratching the monolayer, and the rate of wound closure is measured over time . Researchers can manipulate RMND5A expression through overexpression plasmids or targeting miRNAs like miR-590-5p, then quantify changes in migration rates.

For comprehensive investigation, this approach should be complemented with:

• Transwell migration assays to quantify directional cell movement

• Time-lapse microscopy to track individual cell movements and morphological changes

• Molecular pathway analysis examining known migration regulators

• In vivo metastasis models to validate findings in physiological contexts

These combined methodologies provide robust evidence of RMND5A's role in cancer cell migration, potentially revealing therapeutic targets for controlling metastatic spread .

How does RMND5A interact with other components of the CTLH complex?

In higher organisms, RMND5A is a key component of the CTLH protein complex, which is analogous to the Gid-complex in yeast. While the search results don't provide comprehensive details about all interactions, we know that RMND5A functions within this high molecular weight complex . The CTLH complex contains six conserved Gid proteins, with RMND5A being the ortholog of yeast Gid2 . While both RMND5A and RMND5B exist in humans, only RMND5A is part of the CTLH complex, suggesting specific interaction domains that facilitate its incorporation into this complex .

Research methodologies to investigate these interactions would include co-immunoprecipitation experiments followed by mass spectrometry to identify binding partners, yeast two-hybrid screening to detect direct protein-protein interactions, and structural biology approaches to characterize the three-dimensional organization of the complex. Understanding these interactions is crucial for elucidating how RMND5A contributes to the ubiquitin ligase activity of the CTLH complex.

What are the known and predicted substrates of RMND5A's E3 ubiquitin ligase activity?

Currently, the specific substrates targeted by RMND5A for ubiquitination and subsequent degradation remain largely unknown, particularly in the context of cancer and development. In yeast, the Gid-complex (containing the RMND5A ortholog Gid2) regulates the metabolic switch between glycolysis and gluconeogenesis by targeting specific enzymes . In humans, RMND5A has been reported to protect Exportin5 from proteasomal degradation in conjunction with RanBP10 , suggesting a role in regulating the stability of this protein rather than directly targeting it for degradation.

Research approaches to identify RMND5A substrates should include:

• Proteomics analysis comparing protein levels in RMND5A-overexpressing versus depleted cells

• Ubiquitinome analysis to identify differentially ubiquitinated proteins

• Protein stability assays in the presence or absence of RMND5A

• In silico prediction based on recognition motifs and structural characteristics

Identification of RMND5A substrates would significantly advance our understanding of how this E3 ligase contributes to developmental processes and cancer progression.

How does the regulation of RMND5A by microRNAs vary across different tissue contexts?

The regulation of RMND5A by microRNAs appears to be context-dependent. In pancreatic cancer cells, miR-590-5p has been identified as a direct regulator of RMND5A expression . Additionally, human RMND5A expression has been reported to be regulated by miRNA-138 . This suggests that RMND5A expression is subject to complex post-transcriptional regulation by multiple microRNAs.

The tissue-specific expression patterns of these regulatory microRNAs likely contribute to differential regulation of RMND5A across various tissues and disease states. For instance, the effects of miR-590-5p on tumorigenesis are controversial, with studies showing tumor-suppressive functions in some cancers (breast cancer, non-small cell lung cancer, tongue squamous cell carcinoma) and tumor-promoting effects in others (cervical cancer, renal cell carcinoma, endometrial cancer) . This suggests that the regulation and function of RMND5A may be highly dependent on the tissue microenvironment and specific disease context.

What are the consequences of RMND5A suppression on brain development in Xenopus laevis?

Suppression of rmnd5 function in Xenopus laevis results in specific malformations of the forebrain (prosencephalon) and midbrain (mesencephalon) . This suggests that Rmnd5 plays a critical role in proper brain development, potentially by regulating the degradation of specific factors involved in neuronal differentiation or patterning. The strongest expression of rmnd5 in Xenopus laevis is observed in neuronal ectoderm, prospective brain, eyes, and ciliated cells of the skin , consistent with its developmental role in these tissues.

For researchers investigating this phenomenon, it would be important to:

• Characterize the specific neuronal subtypes affected by rmnd5 suppression

• Identify the temporal window during which rmnd5 function is critical

• Determine whether the effects are cell-autonomous or non-cell-autonomous

• Identify potential substrates of Rmnd5 ubiquitin ligase activity in neural tissues

Understanding the molecular mechanisms underlying these developmental defects could provide insights into human neurodevelopmental disorders, particularly given the report of a duplication of the RMND5A gene associated with a giant occipitoparietal meningoencephalocele in a human infant .

How do researchers address contradictory findings about RMND5A function across different model systems?

When confronting contradictory findings about RMND5A function across different model systems, researchers should implement a systematic approach:

First, carefully evaluate experimental conditions that may explain divergent results, including:

• Cell/tissue-specific contexts (RMND5A appears to have different roles in different tissues)

• Developmental timing (particularly important in developmental studies)

• Methodological differences in RMND5A modulation (knockdown vs. knockout)

• Presence of compensatory mechanisms (such as RMND5B in mammals)

Second, perform comparative studies using consistent methodologies across multiple model systems to directly assess functional conservation and divergence.

Third, examine interaction partners and regulatory networks in each model system, as RMND5A may participate in different protein complexes depending on cellular context .

Finally, consider evolutionary adaptations—while RMND5A is conserved, its specific functions may have diversified. The finding that Xenopus Rmnd5 shows greater homology to human RMND5A (94% identity) than to human RMND5B (70% identity) helps explain some functional differences .

This comprehensive approach helps reconcile seemingly contradictory findings and builds a more complete understanding of this multifunctional protein.

What technical approaches can be used to investigate the role of RMND5A in ciliated cells of Xenopus skin?

Given that rmnd5 expression is strong in ciliated cells of Xenopus laevis skin , several specialized technical approaches can be employed to investigate its function in this context:

• High-resolution imaging techniques:

  • Scanning electron microscopy to visualize ciliary structure

  • Live imaging using tagged proteins to monitor ciliary dynamics

  • Super-resolution microscopy to detect protein localization within cilia

• Functional assays:

  • Ciliary beat frequency measurements using high-speed videomicroscopy

  • Fluid flow analysis to assess ciliary function

  • Mucociliary clearance assays to evaluate transport capability

• Molecular tools:

  • Targeted gene editing using CRISPR/Cas9 with electroporation into skin cells

  • Tissue-specific promoters to drive transgene expression in ciliated cells

  • Conditional knockdown approaches to control timing of RMND5A suppression

• Biochemical approaches:

  • Proximity labeling to identify ciliary-specific interaction partners

  • Comparative proteomics of ciliated cells with and without RMND5A

These approaches would help determine whether RMND5A regulates ciliogenesis, ciliary maintenance, or ciliary function, and could provide insights into human ciliopathies, a diverse group of disorders caused by ciliary dysfunction.