Recombinant Mouse RELT-like protein 2 (Rell2)

What is RELL2 and how does it relate to the RELT family of proteins?

RELL2 (Receptor Expressed in Lymphoid Tissues-like 2) is one of three members of the RELT family proteins (RELTfms), along with RELT (TNFRSF19L) and RELL1. While RELT is a member of the Tumor Necrosis Factor Receptor Superfamily (TNFRSF), RELL1 and RELL2 are paralogs that lack the characteristic cysteine-rich domains used to bind TNFSF ligands but share homology with RELT, particularly in their transmembrane and intracellular domains .

The relationship between these three proteins is characterized by:

• 40% amino acid identity between RELL1 and RELL2

• 32% amino acid identity between RELT and RELL1

• 27% amino acid identity between RELT and RELL2

• Strongest homology within the transmembrane intracellular domains (ICDs)

All three proteins can physically interact and co-localize with each other at the plasma membrane, with RELL1 and RELL2 potentially functioning as modulators of RELT signaling .

What is the molecular structure and key features of mouse RELL2?

Mouse RELL2 is a 303 amino acid-long type I transmembrane protein with a molecular weight of approximately 32.4 kDa. Unlike RELT, RELL2 has a relatively short extracellular domain (ECD) and lacks the cysteine-rich domains typically found in TNFRSF members .

Key structural features include:

• Type I transmembrane orientation

• Disordered sequences in the carboxy-terminal tail, suggesting the ability to adopt multiple conformations depending on post-translational modifications or protein interactions

• Absence of extracellular cysteine-rich domains typical of TNFRSF members

• Plasma membrane localization when co-expressed with other RELTfms

When expressed alone, RELL2 predominantly localizes to the plasma membrane, unlike RELT which tends to localize within cytosolic compartments when expressed independently .

What are the expression patterns of RELL2 in mouse tissues?

RELL2 displays a tissue-restricted expression pattern compared to RELL1. According to multiple expression analyses:

• RELL2 mRNA is predominantly expressed in hematopoietic tissues such as thymus and spleen

• High expression is observed in immune-privileged sites including the testes, brain, and placenta

• Significant expression is found in various brain regions (cerebral cortex, medulla)

• Notable expression in endocrine tissues including parathyroid and pituitary glands

• Minimal expression in tissues such as skeletal muscle

This restricted expression pattern suggests specialized functions in immune-related processes and specific regulatory roles in immune-privileged tissues.

What are the recommended approaches for producing recombinant mouse RELL2 for research applications?

Based on established protocols for similar recombinant proteins, the following methodology is recommended:

• Expression System Selection:

  • Mammalian expression systems (HEK293 or CHO cells) are preferred for proper post-translational modifications

  • Baculovirus-insect cell systems (Sf9 or Sf21) can be used for higher yields with near-native glycosylation patterns

• Vector Design:

  • Include a signal peptide for secretion

  • Add a purification tag (His6, Fc-fusion, or FLAG) at the C-terminus to avoid interference with N-terminal folding

  • For soluble recombinant RELL2, exclude the transmembrane domain

• Purification Protocol:

  • Affinity chromatography using anti-tag antibodies or metal affinity resins

  • Size exclusion chromatography to ensure homogeneity

  • Formulation in PBS with optional stabilizers (similar to protocols for RELT)

• Quality Control:

  • SDS-PAGE to verify purity and molecular weight

  • Western blotting to confirm identity

  • Functional validation through binding assays with known interaction partners

How can researchers validate the activity of recombinant mouse RELL2 in experimental settings?

Validation of recombinant RELL2 activity can be performed through multiple functional assays:

• Binding Studies:

  • Co-immunoprecipitation (co-IP) with RELT and RELL1 to confirm protein-protein interactions

  • Surface plasmon resonance to quantify binding affinity with partners

• Cell-Based Functional Assays:

  • MAPK14/p38 cascade activation assessment using phospho-specific antibodies

  • Apoptosis assays (since RELL2 overexpression induces apoptosis)

  • Cell proliferation assays in cancer cell lines

  • Immune cell activation/suppression assays

• Intracellular Signaling:

  • Reporter gene assays for downstream pathways (NF-κB, MAPK)

  • Phosphorylation analysis of known downstream targets

• Comparative Controls:

  • Include other RELTfm proteins as controls

  • Use targeted mutations in functional domains to validate specific activities

A typical validation protocol would include both binding verification and at least one functional assay to ensure bioactivity.

What experimental models are most suitable for studying RELL2 function in vivo?

The following experimental models have proven valuable for studying RELL2 function:

• Mouse Models:

  • Conditional knockout systems targeting RELL2 expression in specific tissues

  • Transgenic overexpression models to study gain-of-function effects

  • Xenograft models using RELL2-manipulated cancer cells to study oncogenic roles

• Cell Line Models:

  • Cancer cell lines with differential RELL2 expression (e.g., pancreatic cancer lines)

  • Immune cell lines to study immunomodulatory functions

  • Cell lines derived from tissues with high endogenous RELL2 expression (lymphocytes, brain-derived cells)

• Ex Vivo Systems:

  • Primary lymphocyte cultures from mouse lymphoid tissues

  • Organoid cultures from tissues with high RELL2 expression

• Experimental Design Considerations:

  • Include appropriate controls (wildtype littermates, scrambled siRNA)

  • Validate knockdown/overexpression efficiency

  • Consider compensatory effects from other RELTfm family members

When selecting a model, researchers should consider the specific aspect of RELL2 biology they aim to study, as different models may be optimal for investigating cancer progression versus immune regulation.

How does RELL2 expression correlate with patient prognosis across different cancer types?

Analysis of RELL2 expression across 33 cancer types from the TCGA database reveals complex and context-dependent associations with patient outcomes:

Cancers where high RELL2 expression predicts poor survival:

• Adrenocortical carcinoma (ACC)

• Cervical squamous cell carcinoma (CESC)

• Glioblastoma multiforme (GBM)

• Kidney chromophobe (KICH)

• Kidney renal clear cell carcinoma (KIRC)

• Pheochromocytoma and paraganglioma (PCPG)

• Thyroid carcinoma (THCA)

• Uterine carcinosarcoma (UCS)

Cancers where high RELL2 expression predicts better survival:

• Pancreatic adenocarcinoma (PAAD)

• Thymoma (THYM)

The relationship between RELL2 expression and various survival metrics varies by cancer type:

These findings suggest RELL2 may function as either a tumor suppressor or oncogene depending on the cancer context, highlighting the need for cancer-specific investigation.

What mechanisms underlie RELL2's role in cancer progression?

Several molecular mechanisms have been identified that explain RELL2's involvement in cancer progression:

• Regulation of Apoptosis:

  • Overexpression of RELL2 induces activation of the MAPK14/p38 cascade and apoptosis

  • In pancreatic ductal adenocarcinoma (PDAC), RELL2 demonstrates anti-oncogenic properties

• Alternative Splicing and Intron Retention:

  • Intron retention (IR) occurs at the fourth intron of RELL2 transcript in gemcitabine-resistant PDAC cells

  • The upstream splicing factor DHX38 regulates RELL2 intron 4 retention

  • When intron 4 is retained, nonsense-mediated mRNA decay (NMD) is triggered, reducing functional RELL2 protein levels

• Immune Microenvironment Modulation:

  • RELL2 expression correlates with immune and stromal scores in multiple cancer types

  • Significant correlation between RELL2 expression and various immune checkpoint genes

  • Positive correlation with immune neoantigens in cervical squamous cell carcinoma (CESC), kidney renal papillary cell carcinoma (KIRP), and skin cutaneous melanoma (SKCM)

• Association with DNA Repair Mechanisms:

  • RELL2 expression positively correlates with mismatch repair (MMR) genes including MLH1, MSH2, and MSH6 in multiple cancers

  • Negative correlation with EpCAM in several cancer types

• Enriched Signaling Pathways:

  • GSEA analysis identified RIG-I-like receptor (RLR) and apical junction pathways as significantly enriched in relation to RELL2 expression

The context-dependent function of RELL2 across different cancers suggests tissue-specific regulatory mechanisms and interaction networks.

How does RELL2 intron retention affect protein function and cancer phenotypes?

Intron retention in RELL2 represents a critical regulatory mechanism with direct implications for cancer progression:

• Mechanism of Intron 4 Retention:

  • DHX38 (DEAH-Box Helicase 38) directly interacts with RELL2 pre-mRNA

  • Reduced DHX38 expression leads to increased retention of RELL2 intron 4

  • The direct interaction between DHX38 and RELL2 was confirmed through RNA immunoprecipitation (RIP-qPCR)

• Consequences of Intron Retention:

  • Introduction of premature termination codons, triggering nonsense-mediated mRNA decay (NMD)

  • Reduced functional RELL2 protein levels

  • Altered RELL2-mediated signaling pathways

  • Inhibition of apoptosis in pancreatic cancer cells

  • Enhanced cancer cell survival and chemoresistance

• Experimental Evidence:

  • Overexpression of DHX38 reduces RELL2 intron 4 retention

  • Knockdown of RELL2 intron 4 impairs the effect of DHX38 overexpression

  • Functional assays demonstrated that RELL2 plays an anti-oncogenic role in PDAC through:

    • Reduced cell proliferation

    • Enhanced gemcitabine cytotoxicity

    • Increased apoptosis

• Clinical Implications:

  • RELL2 intron 4 retention may serve as a biomarker for chemoresistance

  • Targeting the DHX38-RELL2 splicing axis could represent a novel therapeutic approach for PDAC

  • The mechanism may be relevant in other cancer types where RELL2 demonstrates tumor-suppressive functions

This regulatory mechanism highlights the importance of alternative splicing in modulating RELL2 function in cancer contexts and offers potential diagnostic and therapeutic applications.

How does RELL2 contribute to immune cell function and immune response regulation?

RELL2's role in immune regulation can be understood through several key mechanisms:

• Expression in Immune Tissues:

  • Predominantly expressed in hematopoietic tissues (thymus, spleen)

  • Present in peripheral blood leukocytes (PBLs)

  • Expressed in immune-privileged sites (brain, testes, placenta)

• Correlation with Immune Cell Infiltration:

  • Significant correlations between RELL2 expression and six types of immune infiltrating cells:

    • Macrophages

    • Dendritic cells (DCs)

    • Neutrophils

    • B cells

    • CD8+ T cells

    • CD4+ T cells

• Association with Immune Checkpoint Regulation:

  • Significant correlation between RELL2 expression and multiple immune checkpoint genes

  • Particularly strong associations in kidney chromophobe (KICH), kidney renal clear cell carcinoma (KIRC), and thymoma (THYM)

• Interaction with the RELT Signaling Pathway:

  • Co-expression and physical interaction with RELT

  • Potential modulation of RELT-mediated immune signaling

  • RELT has been shown to both activate T cells and promote an immunosuppressive environment in different contexts

• Correlation with Immune Neoantigens:

  • Positive correlation with neoantigen counts in several cancer types

  • Potential role in neoantigen recognition or presentation

These findings suggest RELL2 may play a multifaceted role in immune regulation, potentially influencing both immune activation and suppression depending on the cellular context.

What experimental approaches can be used to investigate RELL2's interactions with immune checkpoints?

To investigate RELL2's interactions with immune checkpoints, researchers can employ the following methodological approaches:

• Co-expression and Correlation Analysis:

  • RNA-seq or qPCR analysis of RELL2 and immune checkpoint gene expression in immune cells or tumor samples

  • Single-cell RNA sequencing to identify cell type-specific co-expression patterns

  • Correlation analysis using databases like TCGA, GTEx, and TIMER

• Protein-Protein Interaction Studies:

  • Co-immunoprecipitation (Co-IP) to detect physical interactions between RELL2 and immune checkpoint proteins

  • Proximity ligation assay (PLA) to visualize interactions in situ

  • FRET or BRET assays to confirm direct interactions in living cells

  • Yeast two-hybrid screening to identify novel interaction partners

• Functional Impact Analysis:

  • CRISPR-Cas9 knockout or siRNA knockdown of RELL2 in immune cells

  • Flow cytometry to measure changes in immune checkpoint expression

  • T cell activation assays (measuring IL-2 production, proliferation)

  • Mixed lymphocyte reactions to assess T cell responses

  • Checkpoint blockade efficacy testing in RELL2-manipulated models

• In Vivo Immune Response Assessment:

  • Mouse models with conditional RELL2 knockout in specific immune cell populations

  • Tumor challenge studies with immune checkpoint blockade therapies

  • Immune infiltration analysis by flow cytometry or mass cytometry

  • Cytokine profiling in tumor microenvironment

  • Adoptive transfer experiments with RELL2-modified immune cells

• Signaling Pathway Analysis:

  • Phospho-flow cytometry to assess activation of immune signaling pathways

  • Western blotting for key signaling nodes (NF-κB, MAPK pathways)

  • Reporter assays for transcription factor activation

  • Transcriptomic analysis after RELL2 manipulation in immune cells

These approaches should be combined to build a comprehensive understanding of RELL2's role in immune checkpoint regulation.

How does the immune microenvironment correlate with RELL2 expression in different cancer types?

RELL2 expression demonstrates cancer type-specific correlations with the immune microenvironment:

Correlation with Immune and Stromal Scores:

The ESTIMATE algorithm revealed significant correlations between RELL2 expression and immune/stromal components across cancer types:

Correlation with Specific Immune Components:

These differential correlations suggest RELL2 may exert cancer type-specific effects on the immune microenvironment, potentially contributing to its contrasting roles in patient outcomes across different cancer types .

How can structural analysis of RELL2 inform the development of targeted research tools?

Structural characterization of RELL2 provides critical insights for developing targeted research tools:

• Structural Domains and Their Functions:

  • RELL2 lacks the cysteine-rich domains present in RELT

  • Contains disordered sequences in the carboxy-terminal tail

  • Transmembrane domain crucial for membrane localization

  • No death domains typical of apoptosis-inducing TNFRSF members

• Epitope Mapping for Antibody Development:

  • Target unique extracellular epitopes to distinguish from RELL1 and RELT

  • Design antibodies against predicted surface-exposed regions

  • Avoid disordered regions that may present conformational variability

  • Consider species conservation for cross-reactive antibodies

• Structural Considerations for Recombinant Protein Design:

  • Include critical domains for proper folding and stability

  • Preserve regions necessary for interaction with RELT and RELL1

  • Strategic tag placement to avoid interference with functional domains

  • Consider soluble variants lacking the transmembrane domain

• Structure-Based Small Molecule Development:

  • Identify potential binding pockets for small molecule modulators

  • Focus on regions involved in protein-protein interactions

  • Target domains involved in signaling pathway activation

  • Develop compounds that specifically modulate RELL2 versus other RELTfms

• CRISPR-Based Tools:

  • Design guide RNAs targeting conserved exons

  • Consider targeting regions that would not affect RELL1 expression

  • Develop CRISPR activation or inhibition systems targeting RELL2 promoter regions

  • Create domain-specific knockout strategies

Understanding RELL2's structural properties enables the development of more specific and effective research tools to investigate its unique functions distinct from other RELTfm family members.

What are the current technical challenges in studying RELL2 function, and how can they be addressed?

Researchers face several technical challenges when studying RELL2 function:

• Functional Redundancy with Other RELTfms:

  • Challenge: RELL1 and RELL2 share 40% amino acid identity and may have overlapping functions

  • Solution:

    • Use double knockdown/knockout approaches

    • Develop specific inhibitors that distinguish between family members

    • Employ domain-swapping experiments to identify unique functional regions

• Lack of Identified Ligands or Binding Partners:

  • Challenge: Unlike RELT, specific ligands for RELL2 remain unidentified

  • Solution:

    • Perform unbiased protein-protein interaction screens

    • Use proximity labeling approaches (BioID, APEX)

    • Conduct systematic screening with recombinant protein libraries

• Context-Dependent Functions:

  • Challenge: RELL2 appears to have opposing roles in different cancer types

  • Solution:

    • Use tissue-specific conditional knockout models

    • Study RELL2 in relevant tissue microenvironments

    • Identify tissue-specific binding partners

• Complex Alternative Splicing Regulation:

  • Challenge: Intron retention and alternative splicing complicate functional analysis

  • Solution:

    • Design isoform-specific detection methods

    • Use minigene constructs to study splicing regulation

    • Develop tools to specifically target individual RELL2 isoforms

• Low Endogenous Expression Levels:

  • Challenge: RELL2 has restricted tissue expression, making detection difficult

  • Solution:

    • Use highly sensitive detection methods (digital PCR, RNAscope)

    • Focus on tissues with known high expression

    • Develop knock-in reporter systems for in vivo visualization

• Post-Translational Modifications:

  • Challenge: RELL2 likely undergoes significant post-translational modifications

  • Solution:

    • Use mass spectrometry to map modifications

    • Generate modification-specific antibodies

    • Create mutants to assess functional impacts of modifications

Addressing these challenges requires integrated approaches combining advanced molecular biology techniques with appropriate model systems that recapitulate the physiological context of RELL2 function.

How might RELL2 research contribute to the development of novel therapeutic strategies?

RELL2 research opens several promising avenues for therapeutic development:

• Cancer-Specific Therapeutic Approaches:

  • Pancreatic Cancer: Enhancing RELL2 expression or function could improve chemosensitivity and promote apoptosis

  • Kidney Cancers: Inhibiting RELL2 may improve outcomes in KICH and KIRC where high expression correlates with poor prognosis

  • Targeting RELL2 Splicing: Modulating DHX38 activity to control RELL2 intron retention in chemoresistant cancers

• Immune Checkpoint Modulation:

  • RELL2's correlation with immune checkpoint genes suggests potential for combination therapies

  • Targeting RELL2 could enhance responses to existing immune checkpoint inhibitors

  • Cancer-specific approaches needed given the variable correlation patterns

• Biomarker Development:

  • Prognostic Biomarkers: RELL2 expression levels for patient stratification

  • Predictive Biomarkers: RELL2 intron retention as a marker for chemotherapy resistance

  • Response Biomarkers: Changes in RELL2 splicing as indicators of treatment efficacy

• Targeting Protein-Protein Interactions:

  • Disrupting or enhancing interactions between RELL2 and other RELTfms

  • Modulating RELL2's interaction with components of the MAPK signaling pathway

  • Developing peptide mimetics of key interaction domains

• Novel Delivery Strategies:

  • Exosome-based delivery of RELL2 mRNA or protein to tumor cells

  • Nanoparticle-mediated delivery of RELL2-targeting agents

  • Cell-based therapies using engineered cells with modified RELL2 expression

• Combination Therapeutic Approaches:

  • Combining RELL2-targeting strategies with:

    • Conventional chemotherapies

    • Immune checkpoint inhibitors

    • DNA damage response modulators (given associations with MMR genes)

    • Epigenetic modifiers affecting RELL2 expression

The development of these therapeutic strategies requires further mechanistic understanding of RELL2's context-dependent functions and careful consideration of potential off-target effects given its role in normal tissues.

How does RELL2 compare functionally with RuvBL2 in protein homeostasis and stress response?

While RELL2 and RuvBL2 are distinct proteins with different primary functions, emerging research suggests potential parallel or complementary roles in cellular homeostasis:

RuvBL2 Functions in Protein Homeostasis:

• RuvBL2 is involved in the control of protein aggregation

• Assists in compartmentalization of misfolded proteins into the aggresome

• Participates in the disaggregation of large insoluble aggregates

• Loss of RuvBL2 prevents proper aggresome formation and accelerates aggregate accumulation

RELL2 Functions in Cellular Homeostasis:

• Overexpression of RELL2 induces apoptosis via MAPK14/p38 cascade activation

• May regulate cellular survival/death decisions in response to stress

• Functions in a network with RELT family proteins to modulate signaling responses

Comparative Analysis:

Potential Research Directions:

• Investigate whether RELL2 influences protein aggregation or clearance pathways

• Examine if RuvBL2 and RELL2 converge on common stress response pathways

• Study their potential coordinated roles in cellular responses to proteotoxic stress

• Explore whether viral proteins interact with both proteins to modulate host responses

While these proteins have distinct primary functions, understanding their potential functional convergence in stress response pathways could reveal novel regulatory mechanisms governing cellular homeostasis.

What are the emerging techniques for studying RELL2's role in alternative splicing regulation?

Cutting-edge approaches for investigating RELL2's alternative splicing regulation include:

• Long-Read Sequencing Technologies:

  • Oxford Nanopore or PacBio sequencing to capture full-length transcripts

  • Direct RNA sequencing to avoid PCR biases and capture native modifications

  • Detection of complex splicing patterns and rare isoforms

• CRISPR-Based Splicing Modulators:

  • CRISPR-directed RNA targeting systems (Cas13)

  • Base editing of splice regulatory elements

  • Prime editing to modify splice sites with minimal off-target effects

  • CRISPR inhibition targeting splice regulators like DHX38

• Single-Cell Splicing Analysis:

  • Single-cell RNA-seq with specialized computational pipelines for splicing detection

  • SMART-seq protocols to improve full-length transcript coverage

  • Spatial transcriptomics to map splicing patterns within tissue contexts

• In Vivo Splicing Reporters:

  • Bichromatic fluorescent reporters spanning RELL2 intron 4

  • FRET-based splicing sensors

  • Inducible splicing reporter mouse models

• Direct Visualization of Splicing:

  • MS2/MS2CP systems to track nascent transcripts

  • SNAP-tag labeling of splicing factors

  • Live-cell imaging of splicing dynamics

• Structural Analysis of Splicing Complexes:

  • Cryo-EM of DHX38-RELL2 pre-mRNA complexes

  • Hydrogen-deuterium exchange mass spectrometry

  • Cross-linking mass spectrometry to map interaction surfaces

• High-Throughput Splicing Modifier Screens:

  • CRISPR screens targeting splicing regulators

  • Small molecule libraries to identify modulators of RELL2 splicing

  • Antisense oligonucleotide screens to modulate specific splice events

These emerging technologies enable more comprehensive investigation of the complex splicing regulation of RELL2, particularly the clinically relevant retention of intron 4 that impacts cancer progression and chemoresistance .

How might evolutionary analysis of RELL2 inform our understanding of its functional significance?

Evolutionary analysis of RELL2 can provide valuable insights into its functional significance:

• Conservation Across Species:

  • Identification of highly conserved domains suggests functional importance

  • Examination of RELL2 orthologs across mammals, vertebrates, and other taxa

  • Analysis of selection pressures on different protein domains

• Evolutionary Relationship with RELT Family:

  • Understanding the evolutionary origin of the RELT family

  • Determining when RELL1 and RELL2 diverged from RELT

  • Comparing functions of RELT family proteins across species

• Species-Specific Adaptations:

  • Identification of species-specific variations that might reflect adaptive functions

  • Investigation of lineage-specific expansions or losses

  • Correlation of RELL2 sequence variations with species-specific immune adaptations

• Evolutionary Constraints on Alternative Splicing:

  • Analysis of conservation of splicing regulatory elements around intron 4

  • Comparison of intron retention patterns across species

  • Investigation of the evolutionary history of DHX38-RELL2 regulatory relationship

• Coevolution with Interaction Partners:

  • Identification of coevolving protein families

  • Detection of correlated mutations suggesting functional interactions

  • Analysis of coevolution with immune system components across species

• Methodological Approaches:

  • Phylogenetic analysis of RELL2 sequences across species

  • Synteny analysis to examine genomic context conservation

  • Molecular clock analyses to date gene duplication events

  • Positive selection analysis to identify adaptively evolving sites

  • Ancestral sequence reconstruction to infer functional shifts