NUDCD2 Human

What is the genomic structure and location of human NUDCD2?

NUDCD2 (NudC domain containing 2) is a 4-exon protein-coding gene located at chromosome 5q34 in the human genome . The gene encodes NudC-like protein 2, which plays crucial roles in cellular processes involving microtubule dynamics and cell migration . When designing experiments targeting this gene, researchers should consider its compact structure when developing primers or CRISPR guide RNAs. For expression studies, commercially available validated primers like those from GeneCopoeia (catalog number HQP188861-GC) can be used to measure transcript levels .

What are the primary cellular functions of NUDCD2 protein?

NUDCD2 functions primarily as a co-chaperone with Heat shock protein 90 (Hsp90) to regulate multiple cellular processes . Key functions include:

• Regulation of the LIS1/dynein pathway by stabilizing LIS1 with Hsp90

• Restraining centriole amplification by stabilizing HERC2

• Acting as an autophagy receptor that mediates selective autophagic degradation of CP110 at mother centrioles to promote ciliogenesis

• Regulating sister chromatid cohesion by stabilizing cohesin subunits

These diverse functions highlight NUDCD2's importance in maintaining cellular structural integrity and proper cell division.

How should researchers approach NUDCD2 expression analysis?

When analyzing NUDCD2 expression, researchers should consider:

• Transcript variant specificity: By default, qPCR primers are designed to target specific splice variants. For comprehensive analysis, validate whether you need to measure all or specific variants .

• Reference gene selection: For accurate normalization, use validated housekeeping genes appropriate for your tissue type .

• Technical considerations:

  • RNA quality assessment is critical for reliable results

  • Use sequence-verified primers that span exon junctions

  • Include appropriate positive and negative controls

• Protein detection: Complement RNA expression data with protein analysis using validated antibodies that recognize epitopes preserved in clinical samples.

What syndrome is associated with NUDCD2 deficiency?

Biallelic loss-of-function variants in NUDCD2 are associated with a multiple malformation syndrome characterized by :

• Dysmorphic facial features

• Midline brain hypoplasia

• Hypothyroidism

• Cardiovascular abnormalities (pulmonary and aortic valve stenosis)

• Severe liver dysfunction with cholestasis

• Renal failure

• Profound hypotonia

• Early mortality

Cellular analysis has confirmed the absence of NUDCD2 protein in fibroblasts from affected patients with biallelic loss-of-function variants . This syndrome presents with features resembling a ciliopathy but includes additional complications.

How can researchers confirm pathogenicity of novel NUDCD2 variants?

To confirm pathogenicity of novel NUDCD2 variants, implement a multi-layered approach:

• Genetic analysis:

  • Confirm biallelic inheritance pattern

  • Assess variant frequency in population databases

  • Evaluate conservation of affected amino acids across species

  • Use in silico prediction tools to estimate functional impact

• Functional validation:

  • Generate patient-derived fibroblasts to assess NUDCD2 protein expression

  • Perform rescue experiments with wild-type NUDCD2 in patient cells

  • Evaluate key cellular functions (cilia formation, microtubule dynamics)

  • Create equivalent variants using CRISPR-Cas9 in cellular models

• Clinical correlation:

  • Compare patient phenotype with established NUDCD2-deficiency features

  • Document multi-system involvement (brain, liver, kidney, etc.)

What is the relationship between NUDCD2 deficiency and ciliopathies?

The NUDCD2-related syndrome displays features of ciliopathies but with distinctive characteristics . This relationship can be understood through:

• Molecular mechanisms: NUDCD2 promotes ciliogenesis through selective autophagic degradation of CP110 at mother centrioles . Its absence likely impairs primary cilia formation or function.

• Clinical overlap: Features common to ciliopathies include:

  • Brain developmental abnormalities

  • Renal dysfunction

  • Liver involvement

  • Multi-organ system effects

• Distinct features: The severe cholestasis and early mortality distinguish this condition from many classical ciliopathies.

For research purposes, analyzing cilia structure and function in patient-derived cells can help establish the precise mechanistic relationships between NUDCD2 deficiency and ciliary dysfunction.

What CRISPR-Cas9 strategies are optimal for NUDCD2 functional studies?

For effective CRISPR-Cas9 targeting of NUDCD2:

• Guide RNA selection:

  • Use validated gRNA sequences designed by the Zhang laboratory at the Broad Institute

  • Target critical functional domains or early exons to maximize disruption probability

  • Verify gRNA sequence against your target gene sequence before ordering

• Experimental design recommendations:

  • Order at least two gRNA constructs per experiment to increase success probability

  • Select vectors with appropriate selection markers for your cell type

  • Verify knockout efficiency at both RNA and protein levels

• Delivery considerations:

  • For human cell lines, optimized lipofection or nucleofection protocols typically yield highest efficiency

  • Consider lentiviral delivery for difficult-to-transfect cell types

A complete CRISPR construct should contain all elements required for gRNA expression: U6 promoter, spacer (target) sequence, gRNA scaffold, and terminator .

How should researchers investigate NUDCD2 protein interactions?

To effectively study NUDCD2 protein interactions:

• Co-immunoprecipitation approaches:

  • Use antibodies against endogenous NUDCD2 or epitope-tagged versions

  • Include appropriate controls (IgG, knockout cells)

  • Validate interactions under different cellular conditions (stress, cell cycle phases)

• Proximity-based methods:

  • BioID or TurboID fusion proteins to identify proximal proteins

  • FRET or BRET assays for direct interaction studies

  • Proximity ligation assays in fixed cells to visualize interactions in situ

• Key interaction partners to investigate:

  • Hsp90 co-chaperone complex members

  • LIS1 and dynein complex components

  • HERC2 and related proteins

  • Cohesin subunits

• Functional validation:

  • Assess how NUDCD2 depletion affects stability of interaction partners

  • Determine domains required for specific interactions

  • Evaluate effects of patient-derived mutations on interaction capabilities

What cellular models are most appropriate for NUDCD2 research?

Select cellular models based on specific research questions:

• Patient-derived models:

  • Fibroblasts from affected individuals provide direct disease relevance

  • iPSCs can be differentiated into multiple affected tissues (neural, hepatic, renal)

  • Organoids to model organ-specific manifestations

• Engineered cellular systems:

  • CRISPR-Cas9 knockout lines in relevant cell types

  • Cell lines expressing fluorescently tagged NUDCD2 for live imaging

  • Inducible expression systems to study dosage effects

• Model selection considerations:

  • Ciliated cell types are essential for studying ciliogenesis effects

  • Neural cells for investigating LIS1/dynein pathway disruptions

  • Dividing cells for chromosome cohesion studies

  • Multiple cell types to confirm ubiquitous vs. tissue-specific functions

How do cell cycle dynamics influence NUDCD2 function?

Cell cycle-dependent functions of NUDCD2 require specialized experimental approaches:

• Synchronization techniques:

  • Double thymidine block for G1/S boundary arrest

  • Nocodazole treatment for M-phase arrest

  • Serum starvation for G0/G1 enrichment

• Live cell imaging approaches:

  • Fluorescently tagged NUDCD2 to track localization during cell cycle

  • Correlative light and electron microscopy to link localization with ultrastructure

  • FRAP (Fluorescence Recovery After Photobleaching) to assess protein dynamics

• Cell cycle-specific functions to investigate:

  • Sister chromatid cohesion during mitosis

  • Centriole duplication control

  • Microtubule organization at different cell cycle stages

• Analytical considerations:

  • Single-cell analysis to account for population heterogeneity

  • Quantitative image analysis for subtle localization changes

  • Correlation with cell cycle markers to precisely define timing of events

What are the challenges in distinguishing direct vs. indirect effects of NUDCD2 deficiency?

Distinguishing direct from indirect effects presents several methodological challenges:

• Temporal analysis approaches:

  • Acute vs. chronic depletion using inducible systems

  • Time-course experiments to establish sequence of cellular events

  • Pulse-chase studies to track protein stability effects

• Rescue experiment design:

  • Structure-function analysis with domain mutants

  • Complementation with related family members

  • Targeted rescue of specific pathways

• Pathway dissection strategies:

  • Epistasis experiments with known interactors

  • Selective inhibition of downstream pathways

  • Phosphoproteomic analysis to identify signaling changes

• Integrated multi-omics:

  • Combine transcriptomics, proteomics, and metabolomics

  • Network analysis to identify primary vs. secondary nodes

  • Computational modeling to predict cascade effects

How does NUDCD2 compare functionally with other NudC family proteins?

Understanding NUDCD2 in the context of the NudC family requires comparative approaches:

• Functional redundancy assessment:

  • Single and combined knockdown/knockout of family members

  • Rescue experiments with different family proteins

  • Domain swap experiments to identify functional regions

• Expression and localization comparison:

  • Co-expression analysis across tissues and development

  • Co-localization studies during different cellular processes

  • Quantitative assessment of relative abundance

• Interaction partner analysis:

  • Comparative interactome mapping

  • Competition assays for shared binding partners

  • Structural studies of conserved interaction domains

• Disease phenotype comparison:

  • Analysis of patient cohorts with different NudC family mutations

  • Evaluation of complementary vs. distinct pathological features

  • Tissue-specific manifestations of different family member deficiencies

How should researchers approach humanness perception studies in facial difference research?

When studying humanness perception in facial difference research:

• Experimental design considerations:

  • Use faces displaying direct eye gaze, as this signals social interaction invitation and promotes mind perception

  • Consider that direct eye gaze may counterbalance negative effects of facial differences on mind perception

  • Account for severity of facial differences, as mild forms may alter gaze behavior but be insufficient to affect humanness attribution

• Measurement approaches:

  • The Dual Model Questionnaires based on Haslam et al. can measure humanness-related attributes

  • Use numeric scales (e.g., 1-7) for traits to quantify perceptions

  • Consider both uniquely human (UH) traits and human nature (HN) traits

• Eye-tracking methodology:

  • Track patterns of visual attention to specific facial regions

  • Compare exploration patterns between faces with and without differences

  • Correlate gaze behavior with humanness attributions

• Alternative constructs to consider:

  • Objectification may better correspond to lived experiences than dehumanization

  • Investigate meta-dehumanization (how people with facial differences feel dehumanized)

  • Examine self-dehumanization (perceiving oneself as diminished in humanness)

What anthropomorphism measurement tools are relevant for research?

For studying anthropomorphism in research contexts:

• Validated measurement tools:

  • Individual Differences in Anthropomorphism Questionnaire (IDAQ)

  • Consider factor analysis results showing separate factors for animal stimuli vs. non-animal stimuli (technology and nature)

• Application considerations:

  • Individual differences in anthropomorphism predict degree of moral care and concern toward agents

  • These differences also affect amount of responsibility and trust placed on agents

  • Anthropomorphism influences the extent to which an agent serves as a source of social influence

• Methodological recommendations:

  • Include non-anthropomorphic control items in studies

  • Consider domain-specific anthropomorphism (technological vs. natural)

  • Account for cultural differences in anthropomorphic tendencies