NUDT3 Human

What is NUDT3 and which protein family does it belong to?

NUDT3 is a member of the nucleoside diphosphates linked to moiety-X (NUDIX) hydrolase superfamily of enzymes. This family is highly conserved throughout all species and was originally called the MutT family proteins, as MutT was the founding member. NUDIX proteins contain a characteristic NUDIX box motif (Gx5Ex5[UA]xREx2EExGU), which can vary slightly among family members. NUDT3 functions as a hydrolase enzyme that carries out specific hydrolysis reactions against various substrates . The protein is ubiquitously expressed across tissues, with expression patterns that vary depending on cell type and physiological context .

What are the primary cellular functions of NUDT3?

NUDT3 serves primarily as an mRNA decapping enzyme that modulates the stability of a subset of messenger RNAs in mammalian cells. Through its decapping activity, NUDT3 regulates gene expression post-transcriptionally by influencing mRNA turnover rates. Specifically, NUDT3 has been demonstrated to directly control the stability of mRNAs involved in cellular migration, including integrin β6 (ITGB6) and lipocalin-2 (LCN2), while indirectly affecting expression of other motility-related genes like fibronectin (FN) . Additionally, NUDT3 has phosphatase activity against polyphosphate substrates, suggesting multiple biochemical roles within the cell. Polymorphism of the NUDT3 gene has been implicated in increased BMI and obesity, potentially through these phosphatase activities .

How is NUDT3 expression regulated across human tissues?

NUDT3 demonstrates a ubiquitous expression pattern across tissues, though with variable abundance depending on tissue type. Information regarding tissue-specific expression can be found in dedicated protein atlas resources, which contain RNA sequencing data and protein-level expression profiles across different human tissues . Expression data is typically derived from antibody-based profiling using immunohistochemistry across numerous normal tissue types. The reliability of these expression patterns can be assessed through multiple sources, including RNA-seq data, protein characterization studies, and immunohistochemical data using antibodies with non-overlapping epitopes . Understanding tissue-specific expression patterns provides insight into potential tissue-specific functions of NUDT3.

What enzymatic activities does NUDT3 possess?

NUDT3 exhibits dual enzymatic capabilities that contribute to its diverse cellular functions. Primarily, NUDT3 functions as an mRNA decapping enzyme, hydrolyzing the 5' cap structure of mRNAs which initiates their degradation. This activity has been confirmed both in vitro and in cellular contexts, establishing NUDT3 as a bona fide decapping enzyme . Additionally, NUDT3 demonstrates phosphatase activity against polyphosphate substrates, which may contribute to its reported associations with metabolic regulation . The protein shares small molecule dinucleotide hydrolase activity with related family member Nudt4, though unlike Nudt4, NUDT3 possesses the critical decapping functionality that distinguishes its biological role .

How does the mRNA decapping mechanism of NUDT3 function?

NUDT3's decapping activity involves the hydrolysis of the cap structure at the 5' end of mRNAs, which typically protects transcripts from degradation. The catalytic mechanism depends on specific glutamic acid residues within the NUDIX domain that coordinate metal ions essential for the hydrolysis reaction. Mutation of these critical glutamic acid residues to glutamine (referred to as the EE/QQ mutant) abolishes NUDT3's decapping activity while preserving protein structure . This catalytically inactive mutant has been instrumental in demonstrating that the observed effects on mRNA stability and subsequent cellular phenotypes are specifically due to NUDT3's enzymatic decapping function rather than structural or scaffolding roles of the protein .

What are the known mRNA targets of NUDT3 decapping activity?

Research has identified several direct mRNA targets of NUDT3 decapping activity through genome-wide RNA-seq analysis comparing control cells to those with Nudt3 knockdown. Notable direct targets include:

• Integrin β6 (ITGB6) - Stability increases upon NUDT3 depletion, with corresponding increased protein expression

• Lipocalin-2 (LCN2) - Also shows increased stability in NUDT3-deficient cells

Other transcripts like fibronectin (FN) and S100A8 show altered steady-state levels but not altered stability upon NUDT3 knockdown, indicating they are indirectly regulated by NUDT3 . The specificity of these effects was confirmed by complementation studies, where wild-type NUDT3 but not catalytically inactive mutants restored normal mRNA levels and stability .

How does NUDT3 regulate cell migration at the molecular level?

NUDT3 regulates cell migration through its decapping activity on specific mRNAs involved in cellular motility. When NUDT3 is knocked down in MCF-7 breast cancer cells, there is a significant increase in cell migratory capacity, characterized by enhanced lamellipodia/filopodia extensions and increased wound closure in scratch-wound healing assays . This regulation occurs through two primary mechanisms:

• Direct stabilization of motility-related mRNAs: NUDT3 directly targets and destabilizes mRNAs like ITGB6, which encodes integrin β6, a component of the αvβ6 integrin heterodimer that facilitates cell-ECM interactions .

• Indirect regulation of extracellular matrix components: NUDT3 indirectly regulates the expression of fibronectin (FN), an important stromal extracellular matrix protein that promotes cell adhesion and migration .

Importantly, the enhanced migration phenotype observed in NUDT3-depleted cells is dependent on its decapping activity, as demonstrated by rescue experiments where wild-type NUDT3, but not catalytically inactive mutants, reversed the enhanced migration .

What is the role of the integrin β6-fibronectin axis in NUDT3-mediated cell migration?

NUDT3 controls cell migration through a regulatory axis involving integrin β6 and fibronectin:

• Integrin β6 regulation: NUDT3 directly destabilizes ITGB6 mRNA, which encodes the β6 subunit of the αvβ6 integrin heterodimer. When NUDT3 is depleted, ITGB6 mRNA stability increases, leading to higher expression of integrin β6 protein .

• Fibronectin connection: Simultaneously, NUDT3 knockdown leads to increased fibronectin mRNA and secreted fibronectin protein levels. Fibronectin is a key extracellular matrix ligand for integrin αvβ6 .

• Functional significance: Neutralizing antibodies against αvβ6 (antibody 10D5) eliminated the enhanced migration of NUDT3-knockdown cells, demonstrating that this integrin is necessary for the observed phenotype. Similarly, simultaneous knockdown of both fibronectin and NUDT3 prevented the increased motility seen with NUDT3 depletion alone .

This evidence establishes that NUDT3 regulates cell migration through coordinated control of both a cell surface receptor (integrin αvβ6) and its extracellular ligand (fibronectin), creating a regulatory circuit that modulates cell-ECM communication and motility .

What are effective methods for modulating NUDT3 expression in experimental systems?

Based on published research methodologies, several approaches have proven effective for modulating NUDT3 expression:

• RNA interference: Short hairpin RNA (shRNA) directed against NUDT3 has been successfully employed to achieve approximately 80% reduction in NUDT3 mRNA with corresponding significant reduction in protein levels .

• Complementation studies: Expression of shRNA-resistant wild-type or mutant NUDT3 variants can be used to validate knockdown specificity and perform structure-function analyses. This approach has been particularly valuable in distinguishing between catalytic and non-catalytic functions by complementing with the catalytically inactive EE/QQ mutant .

• Expression level considerations: When performing complementation studies, it's important to achieve expression levels comparable to endogenous NUDT3 to avoid overexpression artifacts. Published studies have demonstrated successful complementation with exogenous proteins expressed at levels comparable to endogenous NUDT3 .

These approaches provide powerful tools for investigating NUDT3 function in cellular contexts and establishing causality in observed phenotypes.

How can the decapping activity of NUDT3 be assessed in cellular contexts?

Assessing NUDT3's decapping activity in cells requires approaches that distinguish direct from indirect effects on mRNA stability:

• mRNA stability measurements: Transcriptional inhibition using actinomycin D followed by quantification of target mRNA decay over time has proven effective for determining if NUDT3 directly affects mRNA stability .

• Rescue experiments: Complementing NUDT3-deficient cells with wild-type versus catalytically inactive mutants (e.g., the EE/QQ mutant) can establish whether observed effects depend specifically on decapping activity .

• Direct versus indirect targets: Some transcripts (like ITGB6 and LCN2) show altered stability upon NUDT3 modulation, identifying them as direct targets, while others (like FN and S100A8) show altered steady-state levels without stability changes, indicating indirect regulation .

• Specificity controls: Including related family members that lack decapping activity (like NUDT4) helps establish that observed effects are specific to NUDT3's decapping function rather than other hydrolase activities shared among family members .

How might NUDT3 contribute to cancer progression?

NUDT3's demonstrated role in regulating cell migration suggests potential involvement in cancer progression, particularly metastasis:

• Migration regulation: NUDT3 knockdown significantly enhances cell motility in MCF-7 breast cancer cells through mechanisms involving integrin β6 and fibronectin .

• ECM interaction: By regulating the integrin β6-fibronectin axis, NUDT3 controls how cancer cells interact with their extracellular environment, a critical factor in invasion and metastasis .

• Expression context: While αvβ6 expression is minimal to negligible in normal epithelium, it becomes highly upregulated in neoplastic epithelial cells, suggesting contextual importance in cancer progression .

The regulation of specific mRNAs involved in cellular motility positions NUDT3 as a potential modulator of metastatic capacity, though additional studies across diverse cancer models would further clarify its significance in oncogenic processes.