Recombinant Human Nuclear fragile X mental retardation-interacting protein 2 (NUFIP2)

What is the molecular weight and structure of human NUFIP2?

NUFIP2 is a protein with a calculated molecular weight of approximately 76 kDa, although it is typically observed at 76-82 kDa in experimental conditions. It consists of 695 amino acids and functions as a ribonucleoprotein . The protein contains RNA-binding domains that enable its interaction with various RNA molecules within the cell. Although named "nuclear" FMRP interacting protein 2, studies have shown that NUFIP2 is predominantly localized in the cytoplasm throughout all phases of the cell cycle, contrary to earlier beliefs about its nucleo-cytoplasmic relocalization .

What are the primary cellular functions of NUFIP2?

NUFIP2 serves multiple functions in cellular processes:

• It acts as a ribonucleoprotein large subunit assembly factor, ensuring accurate and efficient assembly of ribosomal subunits, thereby influencing protein synthesis and cellular growth .

• It functions as an RNA-binding protein that participates in post-transcriptional regulation of gene expression .

• It serves as a cofactor for Roquin-mediated mRNA decay, contributing to target mRNA recognition and regulation .

• It participates in stress granule formation during cellular stress responses .

What antibodies are available for NUFIP2 detection and what applications are they validated for?

Several commercially available antibodies have been validated for NUFIP2 detection:

Example 1: Proteintech Antibody (17752-1-AP)

Example 2: Abcam Antibody (ab221639)

• Validated for ICC/IF with human samples

• Tested on PFA-fixed, Triton X-100 permeabilized Caco-2 cells at 4 μg/ml

For detection of endogenous NUFIP2, researchers have established monoclonal antibodies such as 23G8 for western blotting and immunoprecipitation, and 14G9 for intracellular staining and flow cytometry .

How can I study NUFIP2's RNA-binding properties experimentally?

To investigate NUFIP2's RNA-binding properties, researchers can employ several methodologies:

• RNA Immunoprecipitation (RIP): Immunoprecipitate NUFIP2 using validated antibodies and analyze associated RNAs by qPCR or sequencing.

• iCLIP-seq (individual-nucleotide resolution Cross-Linking and ImmunoPrecipitation followed by sequencing): This technique provides high-resolution mapping of protein-RNA interactions. Studies using similar RNA-binding proteins have identified A/U-rich sequences as binding motifs .

• Co-immunoprecipitation with RNase treatment: To distinguish between direct protein-protein interactions versus RNA-mediated interactions. For example, overexpressed NUFIP2 co-immunoprecipitated with GFP-Roquin-1 from HEK293T cell lysates in an RNase-insensitive manner, indicating direct protein interaction independent of RNA .

• In vitro binding assays: Using recombinant purified proteins and synthesized RNA molecules to assess direct binding and determine binding affinity.

How does NUFIP2 contribute to stress granule formation?

NUFIP2 plays a significant role in stress granule biology:

What is known about NUFIP2's involvement in mRNA regulation and decay?

NUFIP2 plays a crucial role in mRNA regulation through several mechanisms:

• Cofactor for Roquin-mediated mRNA decay: NUFIP2 was identified through an RNAi screen as one of the strongest cofactors of Roquin-induced mRNA decay, with its knockdown derepressing ICOS more effectively than that of CNOT1 .

• Cooperative binding to mRNA targets: NUFIP2 and Roquin cooperatively bind to tandem stem-loops in the 3'UTRs of target mRNAs such as ICOS and Ox40. Specifically:

  • Post-transcriptional repression of human ICOS by endogenous Roquin proteins requires two neighboring non-canonical stem-loops in the ICOS 3′-UTR.

  • This unconventional cis-element, as well as another tandem loop known to confer Roquin-mediated regulation of the Ox40 3′-UTR, are bound cooperatively by Roquin and NUFIP2 .

• Stabilization by Roquin: NUFIP2 is destabilized in the absence of Roquin, indicating Roquin may protect NUFIP2 from degradation, which in turn enables NUFIP2 to perform its function in mRNA decay .

How does NUFIP2 interact with Roquin proteins and what is the functional significance?

The interaction between NUFIP2 and Roquin proteins has been well-characterized:

• Direct physical interaction: NUFIP2 binds directly and with high affinity to Roquin-1. This interaction occurs through Roquin's amino-terminus (aa 1–509), which harbors the RING finger and ROQ domain, rather than through the carboxy-terminal sequences that were previously proposed to mediate protein-protein interactions .

• RNase-insensitive binding: The interaction between NUFIP2 and Roquin-1 is resistant to RNase treatment, confirming it is a direct protein-protein interaction rather than being mediated through RNA .

• Endogenous interaction: Endogenous Nufip2 is strongly enriched in immunoprecipitates of endogenous Roquin from MEF cell lysates using anti-Roquin-1/2–specific antibodies, demonstrating the physiological relevance of this interaction .

• Functional cooperation: NUFIP2 and Roquin cooperatively bind to specific RNA structures, enhancing target recognition and regulation. This cooperation appears specific for certain cis-elements in target mRNAs .

• Stability regulation: Roquin binding stabilizes NUFIP2 in cells, suggesting a regulatory mechanism where Roquin levels influence NUFIP2 abundance and consequently its function in post-transcriptional regulation .

What is known about NUFIP2's interaction with the Fragile X Mental Retardation Protein (FMRP)?

NUFIP2 was originally identified as an FMRP-interacting protein, and research has expanded our understanding of this interaction:

• Interactome studies: NUFIP2 is confirmed as part of the FMRP interactome, alongside other proteins including FXR1P and Caprin-1 .

• Domain-specific interactions: FMRP interacted with 180 proteins in pull-down experiments, with NUFIP2 being among those that interacted with both N and C termini of FMRP .

• Functional networks: The FMRP-NUFIP2 interaction is part of larger networks involved in RNA metabolism, ribonucleoprotein stress granule formation, translation, and other cellular processes, suggesting multifaceted roles beyond their direct interaction .

• Disease relevance: Given FMRP's association with fragile X syndrome, one of the most prevalent inherited intellectual disabilities, the interaction with NUFIP2 may have implications for understanding the molecular mechanisms of this condition .

What is the role of NUFIP2 in neurodegenerative disease pathways?

Emerging research suggests NUFIP2 may be involved in neurodegenerative disease mechanisms:

• Tau protein interaction: Studies have identified NUFIP2 as one of six cytoplasmic RNA-binding proteins that tightly bind tau protein. In research focused on Alzheimer's disease and tauopathies, NUFIP2 was among the proteins (alongside ATXN2L, PABPC1, PABPC4, G3BP2, and MCRIP1) found to interact with tau .

• Stress granule connection: NUFIP2's role in stress granule formation connects to neurodegenerative disease mechanisms, as pathological stress granules are increasingly recognized as contributors to neurodegenerative processes .

• RNA metabolism dysfunction: As an RNA-binding protein involved in various aspects of RNA metabolism, NUFIP2 may contribute to the RNA processing defects observed in multiple neurodegenerative conditions .

How might NUFIP2 be involved in viral infection responses?

Recent research has begun to uncover potential roles for NUFIP2 in viral infection contexts:

• Stress granule modulation: As a component of stress granules, NUFIP2 may participate in cellular responses to viral infection, as stress granules are known to play roles in antiviral defense .

• G3BP interaction network: NUFIP2 is part of the CapGUN complex with G3BP proteins, which are targeted by viral proteins. For example, the SARS-CoV-2 nucleocapsid (N) protein binds G3BP1 and rewires its mRNA-binding profile to suppress the physiological stress response of host cells .

• mRNA decay pathways: Through its role in Roquin-mediated mRNA decay, NUFIP2 may influence the stability of immune-related transcripts during viral infection, potentially affecting the inflammatory response .

What experimental challenges exist in studying NUFIP2's diverse cellular functions?

Researchers face several challenges when investigating NUFIP2:

• Subcellular localization: Despite its name suggesting nuclear localization, NUFIP2 is predominantly cytoplasmic but can relocalize under certain conditions. This dynamic localization requires careful experimental design to track its movement and function in different cellular compartments .

• Functional redundancy: NUFIP2 participates in multiple protein complexes and pathways, potentially with redundant functions. Disentangling specific contributions requires sophisticated genetic manipulation approaches such as:

  • siRNA rescue experiments with siRNA-resistant NUFIP2 cDNA to validate specificity of knockdown phenotypes

  • Domain-specific mutants to dissect which interactions are necessary for specific functions

• Context-dependent interactions: NUFIP2's interactions may vary depending on cell type, stress conditions, and other environmental factors, necessitating studies across multiple systems to develop a comprehensive understanding of its biology.

• Technical considerations: When designing experiments to study NUFIP2, researchers should consider:

  • The appropriate cell fixation methods (e.g., PFA fixation with Triton X-100 permeabilization has been validated for immunofluorescence studies)

  • Antibody specificity and validation across different applications

  • The impact of epitope tags, which might interfere with protein function or localization

What are the emerging techniques that could advance our understanding of NUFIP2?

Several cutting-edge approaches could enhance NUFIP2 research:

• Single-molecule imaging: To track NUFIP2's dynamic interactions with RNA and protein partners in living cells.

• Cryo-EM studies: To determine the structure of NUFIP2 within its various protein complexes, particularly with Roquin and in stress granules.

• CRISPR-Cas9 genome editing: To create precise mutations or tagged versions of endogenous NUFIP2 for studying its function without overexpression artifacts.

• Phase separation assays: Given NUFIP2's involvement in stress granules, which are membrane-less organelles formed through liquid-liquid phase separation, in vitro reconstitution of phase separation behavior could provide insights into its structural contributions.

• Patient-derived iPSCs: Studying NUFIP2 in neurons derived from patients with neurodegenerative diseases could reveal disease-specific alterations in its function or localization.

How can researchers effectively study the competition between different NUFIP2 interaction networks?

To investigate how NUFIP2 balances its multiple interactions and functions:

• Competitive binding assays: In vitro studies using purified components to determine binding affinities and competition between different partners (e.g., Roquin vs. FMRP).

• Proximity labeling approaches: BioID or APEX2 fusion proteins could identify the spatial and temporal dynamics of NUFIP2 interactions under different cellular conditions.

• Quantitative proteomics: SILAC or TMT labeling combined with immunoprecipitation to measure changes in the NUFIP2 interactome under different conditions or following specific perturbations.

• Live-cell FRET sensors: To monitor real-time changes in protein-protein interactions involving NUFIP2 during cellular responses.

• Domain-specific mutations: Creating variants of NUFIP2 that selectively disrupt individual interaction interfaces to assess their contributions to different cellular functions.