GEMIN6 Human

What is GEMIN6 and what cellular components does it interact with?

GEMIN6, also known as Gem-associated protein 6, Gemin-6, or SIP2, is a component of the GEMINS protein family involved in the survival of motor neuron (SMN) complex. This complex plays a crucial role in the cytoplasmic assembly of small nuclear ribonucleoproteins (snRNPs) and is localized to both the cytoplasm and nucleus . The SMN complex is involved in snRNP assembly and mRNA processing, with disruptions in these processes potentially resulting in tumorigenesis .

GEMIN6 interacts with several proteins within the SMN complex, including SMN, GEMIN2, GEMIN3, GEMIN4, GEMIN5, and GEMIN7 . Notably, GEMIN6 does not bind directly to SMN but rather interacts with it through GEMIN7, creating an intricate network of protein interactions essential for proper complex functioning . These interactions are critical for the assembly and activity of the SMN complex.

Structurally, human GEMIN6 is a single, non-glycosylated polypeptide chain containing 167 amino acids with a molecular mass of approximately 21.9 kDa . The protein's structure facilitates its function within the larger SMN complex, contributing to RNA processing mechanisms that are fundamental to cellular operations.

How does GEMIN6 expression differ between normal and cancerous tissues?

Multiple studies have consistently demonstrated that GEMIN6 is significantly overexpressed in cancer tissues compared to their normal counterparts . This differential expression is particularly well-documented in lung adenocarcinoma (LUAD), where both transcriptomic data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases and experimental validation through quantitative RT-PCR confirm higher expression in tumor tissues and cell lines .

The overexpression of GEMIN6 follows a progressive pattern that correlates with disease advancement. Higher GEMIN6 expression is observed in advanced pathologic stages and in tumors with more advanced N (nodal) and T (tumor) stages of LUAD . This pattern suggests that GEMIN6 not only participates in cancer initiation but also contributes significantly to disease progression and potentially metastasis.

Beyond lung cancer, GEMIN6 has been found to be unanimously upregulated across multiple cancer types, indicating a potential common oncogenic mechanism across different malignancies . This widespread overexpression in cancer highlights GEMIN6 as a protein of broad oncological significance rather than a tissue-specific phenomenon.

What methods are commonly used to detect and measure GEMIN6 expression?

Researchers employ several complementary techniques to detect and quantify GEMIN6 expression across experimental contexts:

• Transcriptomic Analysis:

  • Analysis of RNA-seq data from TCGA and GEO databases provides expression profiles across large patient cohorts

  • Quantitative reverse transcription PCR (qRT-PCR) serves as the gold standard for validating expression differences in cell lines and tissue samples

• Protein Detection Methods:

  • Western blotting (immunoblot assays) measures protein levels in tissues and cell lines with high specificity

  • Immunohistochemistry (IHC) allows visualization of spatial protein distribution in tissue sections

  • Recombinant protein production in E. coli systems can provide standards for quantitative analyses

• Epigenetic Analysis:

  • Methylation analysis of the GEMIN6 promoter region helps understand regulatory mechanisms, as hypomethylation correlates with increased expression in tumors

  • Treatment with DNA methyltransferase inhibitors like 5-azacytidine (5-Aza) can experimentally manipulate GEMIN6 expression levels

• Functional Expression Analysis:

  • RNA interference using shRNAs provides tools to knockdown GEMIN6 expression for functional studies

  • Cell proliferation assays (BrdU incorporation, growth curves) and cell cycle analysis using flow cytometry measure the functional consequences of expression changes

These methodological approaches enable comprehensive characterization of GEMIN6 expression patterns and provide the experimental foundation for understanding its biological roles in both normal and pathological contexts.

What is the relationship between GEMIN6 and the SMN complex?

GEMIN6 functions as an integral component of the survival of motor neuron (SMN) complex, which plays a crucial role in the biogenesis of small nuclear ribonucleoproteins (snRNPs) and mRNA processing . Within this complex, GEMIN6 interacts indirectly with SMN through GEMIN7, as it does not bind directly to SMN protein . The complete complex consists of multiple proteins including SMN, GEMIN2, GEMIN3, GEMIN4, GEMIN5, GEMIN6, and GEMIN7, functioning as a coordinated molecular machine .

Research has demonstrated that GEMIN6, along with other Gemins, can modulate both the expression and activity of the SMN complex . When GEMIN6 is knocked down experimentally, there is a measurable decrease in SMN complex formation and disruption in the maturation process of certain mRNAs, while the stability of existing mRNAs remains largely unaffected . This indicates GEMIN6's specific role in complex assembly and RNA processing rather than RNA stability.

The functional significance of this relationship extends to disease mechanisms, as the SMN complex has been implicated in tumorigenesis through its effects on snRNP assembly and mRNA processing . This mechanistic link provides insight into how GEMIN6 dysregulation might contribute to cancer development and progression, highlighting the importance of this protein-complex interaction in both normal cellular function and pathological states.

What molecular mechanisms drive GEMIN6's oncogenic properties?

GEMIN6 exerts its oncogenic effects through multiple interconnected molecular mechanisms that collectively promote cancer development and progression:

• c-Myc Stabilization Pathway:

  • GEMIN6 functions through the GEMIN6/AURKB/c-Myc axis to promote tumor growth

  • GEMIN6 increases Aurora kinase B (AURKB) expression, which then phosphorylates c-Myc at Serine 67

  • This phosphorylation prevents c-Myc degradation via the proteasome pathway, effectively stabilizing c-Myc protein levels

  • The stabilized c-Myc then drives oncogenic programs through its well-established functions in promoting proliferation and metabolic reprogramming

• Cell Cycle Regulation:

  • GEMIN6 knockdown experiments demonstrate cell cycle arrest at G0/G1 phase

  • Key regulators of G0/G1 cell cycle transition (CDK2, CDK4, and CDK6) are significantly reduced upon GEMIN6 inhibition

  • These changes in cell cycle regulatory proteins explain how GEMIN6 promotes unrestricted cell proliferation in cancer contexts

• mRNA Processing and RNP Biogenesis:

  • As part of the SMN complex, GEMIN6 influences RNA processing mechanisms

  • GEMIN6 affects the maturation process of specific mRNAs, including AURKB mRNA

  • This creates a regulatory loop where GEMIN6 controls AURKB expression, which in turn stabilizes c-Myc

• Epigenetic Regulation:

  • Hypomethylation in the GEMIN6 promoter region positively correlates with its increased expression in tumors

  • Experimental treatment with DNA methyltransferase inhibitor 5-azacytidine can reverse decreased expression of GEMIN6, confirming epigenetic control mechanisms

• Coexpression Network:

  • GEMIN6 is coexpressed with several genes including SF3B6, CPSF3, and PSMB3, suggesting coordinated regulatory networks

  • Functional enrichment analysis shows GEMIN6-associated genes are involved in cell cycle, mRNA processing, and energy metabolism pathways

These mechanisms create a multi-level regulatory system through which GEMIN6 promotes cancer cell proliferation, survival, and migration while inhibiting normal cell cycle control mechanisms, ultimately contributing to poor clinical outcomes.

How does GEMIN6 influence immune cell infiltration in tumor microenvironments?

GEMIN6 demonstrates a significant negative correlation with immune cell infiltration in lung adenocarcinoma (LUAD), suggesting an important role in modulating the tumor immune microenvironment . This relationship has several important dimensions:

• Negative Correlation with Immune Infiltrates:

  • Studies using single-sample Gene Set Enrichment Analysis (ssGSEA) and Spearman correlation analysis have revealed that GEMIN6 expression is inversely related to immune cell infiltration in LUAD tumors

  • This negative relationship appears consistent across multiple immune cell types, indicating a broad immunosuppressive effect rather than selectivity for specific immune populations

• Mechanistic Implications:

  • While the precise mechanisms remain under investigation, GEMIN6's involvement in RNA processing may affect genes involved in immune recruitment or function

  • Its connection to c-Myc stabilization is noteworthy, as c-Myc is known to influence immune evasion mechanisms in cancer

• Clinical Significance:

  • The negative association between GEMIN6 expression and immune cell infiltration provides a potential explanation for the poor prognosis observed in patients with high GEMIN6 expression

  • Tumors with elevated GEMIN6 levels may create an immunosuppressive microenvironment that helps cancer cells evade immune detection and destruction

  • This relationship suggests that GEMIN6 status might affect patient responses to immunotherapy

• Potential Therapeutic Relevance:

  • The immunosuppressive correlation of GEMIN6 suggests that targeting this protein might potentially enhance immune surveillance

  • Combination approaches targeting both GEMIN6 and immune checkpoints could represent a strategy for overcoming immune evasion in tumors

This relationship between GEMIN6 and immune cell infiltration represents an important aspect of GEMIN6 biology with implications for both understanding cancer progression and developing therapeutic strategies that consider the tumor immune microenvironment.

Which signaling pathways are impacted by GEMIN6 expression?

GEMIN6 influences multiple signaling pathways that collectively contribute to its role in cancer development and progression:

• c-Myc Signaling Pathway:

  • GEMIN6 is significantly involved in c-Myc related signaling pathways, but notably not E2F pathways

  • It regulates c-Myc protein stability through AURKB-mediated phosphorylation at Serine 67, creating a post-translational regulatory mechanism

  • This stabilization activates downstream c-Myc target genes that promote proliferation and oncogenesis

• Cell Cycle Regulatory Networks:

  • Functional enrichment analysis demonstrates that GEMIN6-associated genes are significantly enriched in cell cycle pathways

  • GEMIN6 knockdown reduces expression of key cell cycle regulators including CDK2, CDK4, and CDK6

  • This regulation leads to G0/G1 cell cycle arrest and reduced proliferation when GEMIN6 is inhibited

• mRNA Processing Pathways:

  • GEMIN6 regulates genes involved in mRNA processing through its role in the SMN complex

  • This affects the maturation of various RNAs involved in cellular processes, creating broad downstream effects on gene expression programs

  • The regulation of AURKB mRNA processing by GEMIN6 establishes a key mechanistic link to c-Myc stabilization

• Energy Metabolism Pathways:

  • GEMIN6-associated genes show significant enrichment in pathways related to energy metabolism

  • This connection suggests GEMIN6 may influence metabolic reprogramming, which is recognized as a hallmark of cancer

• Potential Neurotransmitter Pathway Connections:

  • Research has identified interactions between GEMIN6 and pathways targeted by sertindole, an antipsychotic drug

  • Sertindole, which targets dopamine D2 receptors, serotonin 5HT2A receptors, and α1-adrenoceptors, decreases GEMIN6 expression

  • This finding suggests potential crosstalk between GEMIN6 and neurotransmitter signaling pathways that could be therapeutically exploited

The multifaceted influence of GEMIN6 across these diverse signaling networks highlights its potential as a central regulator of cancer cell behavior and underscores its promise as a therapeutic target.

How does GEMIN6 affect cell cycle regulation and mRNA processing?

GEMIN6 plays dual roles in cell cycle regulation and mRNA processing, with interconnected effects that collectively promote cancer cell proliferation:

• Cell Cycle Regulation:

  • Experimental GEMIN6 knockdown demonstrates that it arrests the cell cycle at G0/G1 phase, preventing progression to S phase

  • Key G0/G1 transition regulators (CDK2, CDK4, and CDK6) are significantly downregulated when GEMIN6 is inhibited

  • BrdU incorporation assays confirm decreased DNA synthesis and cell proliferation following GEMIN6 knockdown

  • Colony formation capacity is markedly impaired when GEMIN6 expression is reduced

  • These effects can be partially rescued by c-Myc overexpression, establishing c-Myc as a critical downstream effector

• mRNA Processing Mechanisms:

  • As part of the SMN complex, GEMIN6 participates in the biogenesis of small nuclear ribonucleoproteins (snRNPs)

  • These snRNPs are essential components of the spliceosome that processes pre-mRNA into mature mRNA

  • GEMIN6 knockdown impairs SMN complex formation and disrupts the maturation process of certain mRNAs

  • Specifically, GEMIN6 influences the processing of AURKB mRNA, but not its stability, creating a specific regulatory pathway

  • This establishes a mechanistic cascade where GEMIN6 regulates AURKB mRNA processing, leading to increased AURKB protein, which then stabilizes c-Myc to promote cell cycle progression

• Integrated Regulatory Network:

  • Functional enrichment analysis reveals GEMIN6-associated genes are simultaneously involved in both cell cycle regulation and mRNA processing pathways

  • This dual involvement suggests GEMIN6 coordinates these processes through its influence on RNA processing machinery

  • Heat map analysis has revealed the top 20 coexpressed genes with GEMIN6, including SF3B6, CPSF3, and PSMB3, further supporting its role in coordinated gene expression programs

This dual functionality positions GEMIN6 as a unique regulatory node that connects RNA processing mechanisms to cell cycle control, providing multiple potential intervention points for therapeutic development and explaining its profound effects on cellular proliferation and tumor growth.

What are the optimal protocols for expressing and purifying recombinant GEMIN6?

The expression and purification of recombinant GEMIN6 human protein requires specific optimization for successful experimental outcomes:

• Expression System Selection:

  • E. coli represents the preferred expression system for human GEMIN6 recombinant protein

  • The recombinant protein is typically produced as a single, non-glycosylated polypeptide chain containing the 167 amino acids of the native sequence

  • A 23 amino acid His-tag is commonly added to the N-terminus to facilitate purification, resulting in a fusion protein with a molecular mass of 21.9kDa

• Optimal Construct Design:

  • The recommended construct includes the full GEMIN6 sequence (amino acids 1-167) with an N-terminal His-tag

  • The complete amino acid sequence should match: MGSSHHHHHH SSGLVPRGSH MGSMSEWMKK GPLEWQDYIY KEVRVTASEK NEYKGWVLTT DPVSANIVLV NFLEDGSMSV TGIMGHAVQT VETMNEGDHR VREKLMHLFT SGDCKAYSPE DLEERKNSLK KWLEKNHIPI TEQGDAPRTL CVAGVLTIDP PYGPENCSSS NEIILSRVQD LIEGHLTASQ

  • This construct design balances protein expression efficiency with functional activity

• Purification Protocol:

  • Proprietary chromatographic techniques are typically employed for purification

  • The purification process should achieve greater than 90% purity as determined by SDS-PAGE analysis

  • The final product should appear as a sterile filtered, colorless solution

• Buffer Optimization:

  • The optimal formulation includes 20mM Tris-HCl buffer (pH 8.0), 0.15M NaCl, 30% glycerol and 1mM DTT

  • The recommended protein concentration is 0.5mg/ml for most applications

  • This buffer composition maintains protein stability while preserving functional activity

• Storage Conditions:

  • For short-term use (2-4 weeks), store at 4°C

  • For longer periods, store frozen at -20°C

  • For long-term storage, addition of a carrier protein (0.1% HSA or BSA) is recommended to maintain stability

  • Multiple freeze-thaw cycles should be avoided to prevent protein degradation or aggregation

These optimized conditions ensure production of high-quality recombinant GEMIN6 protein suitable for various experimental applications, including antibody production, protein-protein interaction studies, functional assays, and structural analyses.

What assays can measure GEMIN6 functional activity?

Several complementary assays can be employed to measure different aspects of GEMIN6 functional activity across molecular, cellular, and in vivo contexts:

• SMN Complex Assembly Assays:

  • Co-immunoprecipitation (Co-IP) to assess GEMIN6 interaction with other SMN complex components

  • Size exclusion chromatography to analyze complex formation and integrity

  • These approaches determine whether GEMIN6 properly incorporates into the physiologically relevant SMN complex

• RNA Processing and Splicing Assays:

  • In vitro snRNP assembly assays to measure the activity of the SMN complex containing GEMIN6

  • RT-PCR analysis of splicing products to assess the impact of GEMIN6 on mRNA maturation

  • Analysis of the maturation process of specific mRNAs, such as AURKB, to evaluate GEMIN6's specific effects on RNA processing

• Cellular Proliferation and Cell Cycle Assays:

  • Growth curve analysis to measure proliferation over time following GEMIN6 modulation

  • BrdU incorporation assays to quantify DNA synthesis rates and S-phase entry

  • Colony formation assays to assess long-term proliferative capacity and clonogenic potential

  • Flow cytometry with propidium iodide staining for cell cycle distribution analysis

• Migration and Invasion Assays:

  • Transwell migration assays to quantify cell motility following GEMIN6 manipulation

  • Wound healing (scratch) assays to measure collective cell migration

  • Invasion assays using Matrigel-coated chambers to assess metastatic potential

• Protein Stability and Signaling Pathway Assays:

  • Western blotting to measure levels of c-Myc and phosphorylated c-Myc at Serine 67

  • Cycloheximide chase assays to determine protein half-life and stability following GEMIN6 modulation

  • Analysis of AURKB levels and activity as a mediator of GEMIN6's effects

• In Vivo Functional Analysis:

  • Xenograft tumor models to assess tumor growth, volume, and weight following GEMIN6 manipulation

  • Immunohistochemistry for GEMIN6, c-Myc, and proliferation markers like Ki67 in tumor sections

  • Metastasis assays in animal models to evaluate effects on cancer dissemination

These diverse experimental approaches provide complementary insights into GEMIN6 function, from molecular mechanisms to cellular phenotypes and in vivo consequences, allowing comprehensive functional characterization of this protein in both normal and disease contexts.

What experimental models are best suited for studying GEMIN6 in vivo?

Several experimental models have proven effective for studying GEMIN6 function in vivo, each offering specific advantages for different research questions:

• Xenograft Mouse Models:

  • LUAD cell lines (A549, H1975) with GEMIN6 knockdown or overexpression xenografted into immunodeficient mice provide direct assessment of GEMIN6's impact on tumor growth

  • These models effectively demonstrate that GEMIN6 inhibition significantly impedes tumor growth, as measured by reduced tumor volume, decreased tumor weight, and diminished expression of proliferation markers

  • Xenograft approaches allow for relatively rapid assessment of therapeutic interventions targeting GEMIN6

• Metastasis Models:

  • Tail vein injection models enable study of lung-to-brain metastasis, a critical aspect of advanced cancer progression

  • These models have revealed GEMIN6's important role in promoting metastatic spread, with GEMIN6 knockdown reducing migration ability both in vitro and in vivo

  • Such models provide insights into the later stages of cancer progression that are difficult to recapitulate in vitro

• Drug Treatment Models:

  • Xenograft models treated with GEMIN6-inhibiting compounds (e.g., sertindole) allow assessment of potential therapeutic agents

  • Such models have demonstrated that sertindole treatment significantly inhibits tumor growth compared to control groups, with decreased Ki67, GEMIN6, and c-Myc immunohistochemical signals

  • These approaches bridge the gap between molecular understanding and potential clinical applications

• Genetic Engineering Approaches:

  • While not explicitly described in the provided references, CRISPR/Cas9-mediated knockout or knockin models could provide valuable insights into GEMIN6's physiological roles

  • Inducible systems would allow temporal control of GEMIN6 expression to distinguish between developmental and functional roles

• Patient-Derived Models:

  • Patient-derived xenografts would maintain the heterogeneity and characteristics of original patient tumors

  • Organoid models derived from normal and cancerous tissues could provide an intermediate complexity between cell culture and in vivo approaches

When selecting an appropriate model, researchers should consider factors including immune system involvement (particularly given GEMIN6's negative correlation with immune cell infiltration), specific GEMIN6-dependent mechanisms being studied, and the translational goals of the research project. The choice of model system should be guided by the specific research question and the aspects of GEMIN6 biology under investigation.

How can researchers effectively manipulate GEMIN6 expression in laboratory settings?

Researchers have employed various strategies to manipulate GEMIN6 expression levels for functional studies and therapeutic development:

• RNA Interference (RNAi) Approaches:

  • Lentiviral shRNA vectors have been successfully used to knock down GEMIN6 transcript levels in cancer cell lines such as A549 and H1975

  • Multiple shRNA sequences targeting different regions of GEMIN6 mRNA should be tested to confirm specificity and rule out off-target effects

  • This approach consistently achieves significant reduction in GEMIN6 expression and is suitable for both in vitro and in vivo studies

• Overexpression Systems:

  • Plasmid vectors containing the GEMIN6 coding sequence can be transfected into cells to achieve overexpression

  • Addition of epitope tags (His, FLAG, etc.) facilitates detection and purification of the expressed protein

  • Overexpression studies can help determine whether GEMIN6 upregulation is sufficient to drive oncogenic phenotypes

• CRISPR/Cas9 Gene Editing:

  • For complete knockout studies or tagging of endogenous GEMIN6

  • This approach allows for more physiological studies compared to overexpression models

  • Can create stable cell lines with GEMIN6 deletion, mutation, or fusion tags

• Pharmacological Manipulation:

  • Sertindole has been identified as a compound that decreases GEMIN6 protein levels to less than 70% of control levels

  • This provides a chemical biology approach that complements genetic manipulation strategies

  • Concentration-dependent effects have been observed, with more dramatic inhibitory effects on tumor cell survival compared to normal cells

• Rescue Experiments:

  • Co-expression of GEMIN6 with downstream effectors like c-Myc or AURKB to validate mechanistic relationships

  • These experiments have demonstrated that AURKB overexpression can reverse GEMIN6 knockdown-reduced cell proliferation and migration abilities

  • Similarly, c-Myc overexpression can overcome the reduced cell proliferation caused by GEMIN6 knockdown

• Epigenetic Modulation:

  • DNA methyltransferase inhibitor 5-azacytidine (5-Aza) can modulate GEMIN6 expression by affecting promoter methylation

  • This approach provides insight into the epigenetic regulation of GEMIN6 and offers an alternative strategy for expression manipulation

These complementary approaches allow researchers to comprehensively study GEMIN6 function through both gain-of-function and loss-of-function strategies, with appropriate controls to ensure specificity and validate mechanisms. The choice of manipulation strategy should be guided by the specific research question, experimental system, and desired outcomes.

How does GEMIN6 expression correlate with clinical outcomes in cancer patients?

GEMIN6 expression demonstrates strong and consistent correlations with clinical outcomes in cancer patients, particularly in lung adenocarcinoma (LUAD):

These consistent correlations across multiple clinical parameters and independent datasets strongly support GEMIN6's utility as both a prognostic biomarker and potential therapeutic target in cancer, particularly LUAD.

What is the current evidence for GEMIN6 as a cancer biomarker?

Substantial evidence supports GEMIN6's potential as a cancer biomarker, particularly for lung adenocarcinoma:

Collectively, these findings establish GEMIN6 as a promising cancer biomarker with robust diagnostic and prognostic capabilities, particularly in LUAD. The strength of evidence suggests GEMIN6 merits further investigation in larger, prospective clinical validation studies to confirm its utility for clinical implementation.

How might targeting GEMIN6 lead to novel therapeutic strategies?

Targeting GEMIN6 presents several promising therapeutic avenues for cancer treatment based on current research findings:

• Direct GEMIN6 Inhibition Strategies:

  • RNA interference approaches (siRNA/shRNA) have demonstrated that GEMIN6 knockdown effectively reduces tumor cell proliferation, migration, and colony formation in vitro

  • GEMIN6 inhibition significantly impedes xenograft tumor growth in vivo, with marked reductions in tumor mass, volume, and weight

  • These preclinical results provide strong proof-of-concept evidence that direct GEMIN6 targeting could be therapeutically effective

• Drug Repurposing: Sertindole:

  • Screening the FDA-Approved Drug Library Mini identified sertindole as a compound that effectively decreases GEMIN6 protein levels

  • Sertindole treatment showed more dramatic inhibitory effects on tumor cell survival compared to normal cells, suggesting a favorable therapeutic window

  • In xenograft models, sertindole treatment significantly reduced tumor growth, with decreased Ki67, GEMIN6, and c-Myc signals confirmed by immunohistochemistry