NUDT21 Human

What is NUDT21 and what is its primary function in human cells?

NUDT21, also known as cleavage and polyadenylation specificity factor subunit 5 (CPSF5), functions as a subunit of the cleavage factor Im (CFIm) complex required for 3′-UTR cleavage and polyadenylation. Its primary role involves sequence-specific recognition of UGUA elements upstream of poly(A) sites (PAS), making it a determinant player in alternative polyadenylation of mRNA transcripts . Through this mechanism, NUDT21 regulates gene expression by influencing 3′-UTR lengths, which affects susceptibility to microRNA-mediated silencing and other post-transcriptional regulatory processes.

How does NUDT21 regulate alternative polyadenylation?

NUDT21 regulates alternative polyadenylation through a specific molecular mechanism:

• It binds to UGUA elements upstream of potential polyadenylation sites

• When bound, it protects proximal PAS from cleavage by the CPSF complex

• This protection leads to the use of distal PAS and generation of longer 3′-UTRs

• Conversely, NUDT21 knockdown leads to increased use of proximal PAS, resulting in shortened 3′-UTRs

For example, in human tendon stem cells, NUDT21 binds to UGUA elements upstream of the proximal PAS in HMGA2 3′-UTR, protecting this site from cleavage. This results in the use of the distal site, generating a longer 3′-UTR containing multiple microRNA binding sites that increase HMGA2's susceptibility to negative regulation by microRNAs like let-7 .

What are the different subunits of the CFIm complex and how do they interact with NUDT21?

The CFIm complex consists of three main subunits that interact in specific ways:

Research on glioblastoma cells has revealed that these proteins form three distinct subcomplexes. NUDT21 plays a critical role in CFIm complex formation and CFIm59 stability, suggesting a hierarchical organization within the complex . This relationship has significant implications for cancer prognosis, as CFIm59 predicts outcomes similar to NUDT21, while CFIm68 does not show the same correlation.

What experimental methods are commonly used to study NUDT21 expression and function?

Researchers employ several methodological approaches to study NUDT21:

• Expression analysis:

  • qRT-PCR for quantifying mRNA levels

  • Immunohistochemistry for protein detection in tissues

• Functional manipulation:

  • siRNA or shRNA transfection for transient knockdown

  • Lentiviral delivery systems for stable NUDT21 knockdown

  • Targeting sequences: shNudt21-1 (5′-GCA CCA GGA TAT GGA CCC ATC ATT T-3′) and shNudt21-2 (5′-TGA ACC TCC TCA GTA TCC ATA TAT T-3′)

• APA pattern analysis:

  • 3′-RACE analysis for identifying polyadenylation sites

  • Poly(A)-Click-Seq (PAC-seq) for genome-wide APA profiling

• Functional assays:

  • Colony formation assays

  • Cell Counting Kit-8 (CCK-8) for proliferation

  • Flow cytometry for cell cycle analysis

  • β-galactosidase staining for senescence assessment

• Interaction studies:

  • STRING web tool for protein-protein interaction prediction

  • PathScan intracellular signaling arrays for pathway analysis

  • Luciferase reporter assays for studying microRNA regulation effects

How is NUDT21 expression typically altered in cancer cells compared to normal cells?

NUDT21 expression shows cancer type-specific alterations:

These differential expression patterns suggest context-dependent roles for NUDT21, potentially serving as an oncogene in some cancers and a tumor suppressor in others, particularly in brain malignancies.

What is the mechanism by which NUDT21 regulates HMGA2 expression and how does this affect stem cell function?

NUDT21 regulates HMGA2 expression through a complex mechanism of alternative polyadenylation:

• The HMGA2 3′-UTR contains:

  • A proximal PAS between the first and second let-7 binding sites

  • A distal PAS downstream of the seventh let-7 binding site

• NUDT21 binds to UGUA elements upstream of the proximal PAS

• This binding protects the proximal site from CPSF-mediated cleavage

• Protection leads to distal PAS usage, generating a longer 3′-UTR

• The longer 3′-UTR contains multiple let-7 binding sites

• This increases microRNA-mediated silencing and reduces HMGA2 expression

In human tendon stem cells (hTSCs), oxidative stress (H₂O₂) increases NUDT21 expression, leading to more HMGA2 transcripts with longer 3′-UTRs. The consequent reduction in HMGA2 expression results in loss of stemness and pluripotency. Conversely, NUDT21 knockdown increases the proportion of HMGA2 transcripts with shorter 3′-UTRs due to proximal PAS selection, allowing HMGA2 to evade let-7 inhibition, maintain higher expression levels, and preserve stemness properties .

How does NUDT21 influence tumor proliferation and apoptosis in glioblastoma?

NUDT21 exhibits context-dependent effects on tumor proliferation and apoptosis:

In glioblastoma (GBM):

• Functions as a tumor suppressor

• Reduced expression associates with worse survival

• NUDT21 depletion causes:

  • Increased proximal PAS usage

  • Global 3′-UTR shortening

  • Enhanced expression of oncogenic proteins

  • Increased cell proliferation

  • Enhanced tumorigenicity

In head and neck squamous cell carcinoma (HNSCC):

• Silencing NUDT21 produces opposite effects:

  • Inhibited proliferation

  • Promoted apoptosis

  • Cell cycle blockade in G2/M phase

  • Suppression of apoptotic proteins

These contrasting effects highlight NUDT21's context-dependent functions in cancer biology, operating through different molecular mechanisms depending on the cellular environment and cancer type.

What are the key downstream targets of NUDT21-regulated alternative polyadenylation in cancer cells?

Several critical downstream targets of NUDT21-regulated APA have been identified:

These targets highlight NUDT21's role in regulating fundamental cellular processes including signaling pathways, stemness, and cell proliferation through alternative polyadenylation mechanisms.

How can NUDT21 expression be leveraged as a prognostic biomarker in different cancer types?

NUDT21 shows significant potential as a prognostic biomarker:

For clinical implementation, standardized assays quantifying NUDT21 expression in patient samples could be developed, potentially combining measurements with key target genes like Pak1 for enhanced accuracy.

What is the relationship between NUDT21 expression and the tumor immune microenvironment?

NUDT21 expression significantly influences the tumor immune microenvironment:

• Correlation with immunosuppressive cells:

  • Strong positive correlation with M2 macrophages in:

    • HNSC, SKCM, TGCT, KIRC, LUSC

  • Positive association with other immunosuppressive cells:

    • Cancer-associated fibroblasts (CAFs)

    • Regulatory T cells (Tregs)

    • Myeloid-derived suppressor cells (MDSCs)

• Negative impact on anti-tumor immunity:

  • Negative correlation with CD8+ T cells and activated NK cells

  • Particularly strong negative correlations with CD8+ T cells in:

    • HNSC, BRCA, KIRC, UCEC

• Immune checkpoint regulation:

  • NUDT21 plays an essential role in the immunosuppressive environment

  • Suppresses immune checkpoint effects in most cancers

These findings suggest NUDT21 may promote an immunosuppressive tumor microenvironment, potentially contributing to immune evasion and tumor progression, with implications for immunotherapy response.

How do experimental approaches for NUDT21 knockdown or overexpression affect cellular phenotypes?

Experimental manipulation of NUDT21 reveals diverse effects on cellular phenotypes:

These varied responses highlight the context-dependent nature of NUDT21 function, where its role shifts depending on cellular context, disease state, and tissue type.

What are the challenges in targeting NUDT21 or its regulated pathways for potential therapeutic interventions?

Targeting NUDT21 for therapy presents several challenges:

• Context-dependent functions:

  • Tumor suppressor in glioblastoma

  • Potential oncogene in head and neck cancers

  • Inhibition might benefit some cancers but worsen others

• Broad downstream effects:

  • As a master regulator of APA, affects numerous targets simultaneously

  • Poly(A)-Click-Seq analysis shows many Ras pathway genes affected

  • Increases risk of off-target effects

• Complex protein interactions:

  • Functions within CFIm complex with CFIm59 and CFIm68

  • CFIm59 stability depends on NUDT21 levels

  • Targeting NUDT21 disrupts multiple protein interactions

• Impact on normal cell function:

  • Essential roles in normal RNA processing

  • Regulates stem cell differentiation and pluripotency

  • Potential for toxicity in normal tissues

• Delivery challenges:

  • Developing drugs to modulate APA is technically challenging

  • Brain tumors require blood-brain barrier penetration

Given these challenges, targeting key downstream effectors of NUDT21, such as Pak1 in glioblastoma , might offer more specific therapeutic opportunities than directly targeting NUDT21 itself.

How does NUDT21 interact with other RNA processing factors to coordinate gene expression?

NUDT21 functions within a complex network of RNA processing factors:

• CFIm complex interactions:

  • Forms three distinct subcomplexes with CFIm59 and CFIm68

  • CFIm59 protein levels depend on NUDT21 expression

  • CFIm68 levels remain independent

• CPSF complex crosstalk:

  • NUDT21 binding to UGUA elements prevents CPSF-mediated pre-mRNA cleavage

  • This crosstalk is crucial for polyadenylation site selection

• Broader protein interaction network:

  • STRING analysis reveals 10 significant protein-protein interactions

  • Key co-expressed genes include DHFR, OGFOD1, RFWD3, SF3B3, and USP10

• Functional pathway integration:

  • GO functional enrichment shows association with:

    • Nucleoplasmic transport

    • Cell cycle activity

  • Suggests integration with broader cellular processes

These interactions position NUDT21 within an interconnected network of RNA processing factors that collectively regulate gene expression through alternative polyadenylation and related mechanisms.

What genomic and epigenetic factors influence NUDT21 expression and function?

Several factors regulate NUDT21 expression and function:

• Cellular stress responses:

  • Oxidative stress (H₂O₂ treatment) increases NUDT21 expression in human tendon stem cells

  • Links stress response pathways to APA regulation

• Cancer-associated pathways:

  • Elevated in altered SWI/SNF complex in Glioma

  • Increased in p53/rb related pathway

  • Critical cancer pathways can modulate NUDT21 levels

• Genomic instability markers:

  • Significant correlation with tumor mutational burden (TMB)

  • Association with microsatellite instability (MSI)

  • Suggests bidirectional relationship between genomic instability and APA

• Epigenetic connections:

  • Strong association with:

    • Genomic stability

    • Mismatch repair genes

    • Tumor stemness

    • RNA methylation

Understanding these regulatory relationships could help identify contexts where NUDT21 function is particularly important and guide strategies to modulate its expression or activity.

How can combined biomarkers (e.g., NUDT21 and PAK1) improve prognostic accuracy in cancer?

Combined biomarker approaches offer enhanced prognostic value:

• NUDT21 and PAK1 in glioblastoma:

  • Combination provides stronger prognostic indication than either alone

  • Captures both the regulator (NUDT21) and downstream target (PAK1)

  • Offers more complete picture of the dysregulated pathway

• Mechanistic basis:

  • PAK1 is directly regulated by NUDT21 through APA

  • Plays significant role in promoting tumorigenicity

  • Biological relationship provides rationale for combined value

• Implementation strategy:

  • Develop multiplex assays for simultaneous measurement

  • Apply machine learning to integrate values into prognostic score

  • Validate in clinical cohorts

• Potential for additional combinations:

  • Other Ras pathway targets identified by Poly(A)-Click-Seq

  • Could create cancer-specific prognostic signatures

  • Opportunity to develop personalized treatment approaches

This strategy of combining master regulators with key downstream targets represents a promising approach for developing more accurate and biologically informative cancer biomarkers.