DDX21 Antibody

What is DDX21 and what are its primary cellular functions?

DDX21, also known as DEAD (Asp-Glu-Ala-Asp) box polypeptide 21, is a 783 amino acid protein that functions as an RNA helicase with essential roles in RNA metabolism. Its primary functions include:

• Unwinding double-stranded RNA and folding single-stranded RNA into secondary structures

• Facilitating ribosome and spliceosome assembly

• Regulating both RNA Polymerase I and II transcription

• Processing ribosomal RNA (rRNA)

• Contributing to innate immune responses against viral infections

• Promoting cell cycle progression

DDX21 is primarily localized in the nucleus, specifically in the nucleolus, where it participates in the multi-protein B-WICH complex. Its activity can be influenced by various post-translational modifications that affect its helicase activity and protein interactions. In certain cellular contexts, it can undergo translocation from the nucleolus to other cellular compartments in response to specific stimuli .

What cellular processes are regulated by DDX21?

DDX21 is involved in regulating multiple cellular processes critical for normal cell function:

DDX21 has been identified as playing crucial roles in cancer progression, particularly in colorectal, breast, and pancreatic cancers. Recent research indicates that DDX21 interacts with WDR5 to promote colorectal cancer cell proliferation by activating CDK1 gene expression and influencing histone modifications (H3K4me3) on the CDK1 promoter . This finding provides new insights into potential therapeutic targets for cancer treatment.

How is DDX21 expression regulated in normal versus disease states?

DDX21 expression varies between normal and disease states, particularly in cancer and viral infections:

In cancer:

During viral infections:

• HCMV infection prevents the normal repression of DDX21 at both protein and mRNA levels

• Virus infection can induce DDX21 translocation from the nucleus to the cytoplasm

• Caspase-dependent cleavage of DDX21 occurs after virus infection, especially with RNA viruses

Research shows that DDX21 undergoes caspase-dependent cleavage at D126 during viral infections, which promotes its translocation from the nucleus to the cytoplasm. This cleaved form of DDX21 negatively regulates interferon beta (IFN-β) signaling by suppressing the formation of the DDX1-DDX21-DHX36 complex, thus modulating the host immune response .

What are the optimal applications for different types of DDX21 antibodies?

Different DDX21 antibodies are optimized for specific experimental applications:

When selecting a DDX21 antibody, researchers should consider:

• The specific cellular compartment being investigated (nucleolar vs. nucleoplasmic vs. cytoplasmic)

• The experimental technique and required sensitivity

• The species of the experimental model

• Whether post-translational modifications or specific DDX21 isoforms are being studied

For example, when studying DDX21 translocation during viral infection, immunofluorescence with antibodies validated for subcellular localization would be most appropriate. For examining protein-protein interactions, antibodies optimized for co-immunoprecipitation would be preferred .

How can I optimize DDX21 antibody use for immunohistochemistry in tissue samples?

For optimal DDX21 immunohistochemical (IHC) staining in tissue samples:

Protocol optimization:

• Tissue preparation: Deparaffinize in xylene and rehydrate in graded alcohols

• Antigen retrieval: Use 0.01 M sodium citrate buffer (pH 6.0) or TE buffer (pH 9.0) in a steamer

• Endogenous peroxidase blocking: Treat with 1% hydrogen peroxide in methanol for 30 minutes

• Background blocking: Pre-incubate in 10% normal FBS for 1 hour

• Primary antibody: Incubate with DDX21 antibody at appropriate dilution (typically 1:50-1:500) at room temperature for 2 hours

• Detection: Follow standard protocol based on DAKO Envision kit using polymer to amplify signals

Critical considerations:

• DDX21 is predominantly nucleolar/nuclear in normal cells but may show altered localization in cancer or infected cells

• For dual staining, select compatible secondary antibodies to avoid cross-reactivity

• Include positive controls (colon cancer tissue, breast cancer tissue) and negative controls (skeletal muscle tissue)

• Confirm antibody specificity through western blot validation before IHC studies

Tissue microarray analysis has shown that DDX21 expression is elevated in breast cancer compared to adjacent normal tissues, with potential prognostic implications . Similar patterns have been observed in colorectal cancer, suggesting broader relevance across multiple cancer types.

What strategies can be used to study DDX21 subcellular localization changes during viral infection?

DDX21 undergoes dynamic subcellular relocalization during viral infection, making it an important aspect of host-pathogen interaction studies:

Methodological approach:

• Immunofluorescence analysis:

  • Fix cells with 4% paraformaldehyde (20 min) and permeabilize with 0.2% Triton X-100 (20 min)

  • Block with 2% BSA (1 hour) followed by overnight antibody incubation at 4°C

  • Use specific cellular markers (nucleolar, nuclear, cytoplasmic) for co-localization studies

  • Analyze using confocal microscopy for precise subcellular localization

• Nucleocytoplasmic separation assay:

  • Fractionate cells into nuclear and cytoplasmic compartments

  • Analyze DDX21 distribution via western blotting

  • Include proper loading controls for each fraction (e.g., lamin for nuclear, GAPDH for cytoplasmic)

• Time-course experiments:

  • Monitor DDX21 localization at different time points post-infection

  • Correlate with viral replication stages and immune response activation

Research has demonstrated that during HCMV infection, DDX21 translocates from the nucleolus to the nucleoplasm . In contrast, PRRSV infection promotes DDX21 translocation from the nucleus to the cytoplasm . For PRRSV, this translocation is independent of DDX21's ATPase, RNA helicase, and foldase activity, highlighting virus-specific mechanisms of DDX21 regulation.

How does DDX21 contribute to cancer progression and what are the implications for cancer research?

DDX21 plays multiple roles in cancer progression through several mechanisms:

Mechanisms of DDX21-mediated cancer progression:

• Cell cycle regulation:

  • Promotes G2/M phase progression by activating CDK1 expression

  • Interacts with WDR5 to enhance H3K4me3 histone marks on cell cycle gene promoters

• Transcriptional regulation:

  • Functions as a cofactor for JUN-activated transcription

  • Required for phosphorylation of JUN at Ser-77, enhancing AP-1 transcription

• RNA metabolism alterations:

  • Affects ribosome biogenesis and translation efficiency in cancer cells

  • Regulates splicing patterns of cancer-related genes

• Epigenetic modulation:

  • Forms complexes with epigenetic regulators like WDR5

  • Contributes to establishment of cancer-specific gene expression patterns

Pan-cancer analysis has revealed that DDX21 is overexpressed in multiple cancer types and correlates with poor prognosis. For example, DDX21 knockdown significantly inhibits colorectal cancer cell proliferation and blocks cell cycle at G2/M phase, suggesting its potential as a therapeutic target .

In pancreatic cancer, DDX21 is upregulated regardless of molecular subtype (classical or basal-like), indicating its fundamental role in PDAC pathogenesis . This consistent overexpression makes DDX21 a promising biomarker for pancreatic cancer detection and prognosis.

What is the role of DDX21 in viral infection and how does it regulate host innate immunity?

DDX21 has complex, sometimes opposing roles in viral infection and innate immunity:

Pro-viral functions:

• Positively regulates replication of certain viruses (e.g., PRRSV, HCMV)

• Cleaved DDX21 suppresses IFN-β signaling pathway

• Required for proper viral late gene transcription in HCMV infection

Anti-viral functions:

• Forms part of DDX1-DDX21-DHX36 complex that senses viral dsRNA

• Binds adaptor protein TRIF to initiate immune signaling

• Inhibits influenza A virus replication (though counteracted by viral NS1 protein)

Regulatory mechanisms:

• Cleavage-dependent regulation:

  • Undergoes caspase-3/6-dependent cleavage at D126 following virus infection

  • Cleaved DDX21 translocates from nucleus to cytoplasm

  • This processed form suppresses the formation of the DDX1-DDX21-DHX36 complex

• R-loop regulation:

  • DDX21 prevents R-loop accumulation on viral late genes

  • Knockdown leads to increased R-loops, impairing viral late gene transcription

  • Overexpression of RNase H rescues this defect, confirming the R-loop mechanism

Experimental evidence shows that DDX21 knockdown significantly reduces vesicular stomatitis virus (VSV) replication but also inhibits interferon production and interferon-stimulated gene expression. This dual effect suggests that DDX21 helps maintain a balance in innate immune responses .

For HCMV, DDX21 mRNA and protein levels are maintained during infection (while they decline in uninfected cells), and its translocation from nucleolus to nucleoplasm is essential for proper viral replication, particularly late gene expression .

How can knockdown and overexpression models be used to study DDX21 function in disease contexts?

Knockdown and overexpression models provide complementary approaches to understand DDX21 function:

Knockdown methodologies:

• siRNA approach:

  • Transient knockdown using specific siRNAs (e.g., siDDX21-3: 5′-CCCATATCTGAAGAAACTATT-3′)

  • Evaluate knockdown efficiency by qRT-PCR and western blotting

  • Best for short-term experiments (3-5 days)

• shRNA approach:

  • Lentiviral delivery of shRNAs for stable knockdown

  • Selection with puromycin for pure populations

  • Suitable for long-term studies and in vivo experiments

• CRISPR/Cas9 gene editing:

  • Creation of DDX21+/- cells with reduced expression

  • More complete and permanent than RNAi approaches

  • Allows for study of partial loss-of-function

Overexpression systems:

• Transient transfection:

  • Plasmid-based expression of wild-type or mutant DDX21

  • Useful for structure-function analyses

  • Enables study of specific domains or post-translational modifications

• Lentiviral overexpression:

  • Stable integration for consistent expression levels

  • Required for long-term studies of DDX21 gain-of-function

  • Example: 513B-DDX21 system used in lung adenocarcinoma studies

Experimental readouts:

• Cell proliferation (CCK8 assay, colony formation)

• Cell cycle analysis (flow cytometry)

• Viral replication (viral titers, viral protein expression)

• Immune signaling (IFN-β luciferase reporter, ISG expression)

• Gene expression (qRT-PCR, RNA-seq)

• Protein-protein interactions (co-IP, proximity ligation)

Research has shown that DDX21 knockdown in lung adenocarcinoma cells significantly inhibits cell proliferation and migration, while overexpression enhances these processes . In viral studies, knockdown and overexpression models have revealed context-dependent roles for DDX21, with discrepancies between these models highlighting the importance of post-translational modifications in regulating DDX21 function .

How can ChIP and DRIP assays be used to investigate DDX21's role in chromatin regulation?

ChIP (Chromatin Immunoprecipitation) and DRIP (DNA-RNA Immunoprecipitation) assays are powerful techniques to investigate DDX21's chromatin functions:

ChIP assay applications:

• Promoter binding analysis:

  • Design walking primers across gene promoters (e.g., CDK1 promoter)

  • Immunoprecipitate DDX21-bound chromatin using specific antibodies

  • Determine enrichment at specific genomic regions via qPCR

  • Example: DDX21 binding to P5 region of CDK1 promoter in colorectal cancer

• Histone modification correlation:

  • Perform parallel ChIP for DDX21 and histone marks (e.g., H3K4me3)

  • Compare enrichment patterns to identify relationships

  • Assess changes in histone marks following DDX21 knockdown/overexpression

  • Research shows DDX21 binding regions correlate with H3K4me3 enrichment

• Complex formation analysis:

  • Use sequential ChIP (Re-ChIP) to identify co-binding partners (e.g., WDR5)

  • Examine recruitment of RNA polymerases and transcription factors

  • Map DDX21-containing complexes genome-wide

DRIP assay applications:

• R-loop detection:

  • Use S9.6 antibody (specific for DNA-RNA hybrids) for immunoprecipitation

  • Measure R-loop accumulation at specific genomic loci

  • Compare R-loop formation in DDX21 knockdown vs. control cells

  • Example: DDX21 knockdown increases R-loops on HCMV late genes

• R-loop resolution mechanisms:

  • RNase H treatment controls to confirm specificity

  • Rescue experiments using RNase H overexpression

  • Time-course analyses to track dynamic changes in R-loop formation

Recent research has demonstrated that DDX21 knockdown leads to increased R-loop accumulation on viral late genes during HCMV infection, impairing their transcription. This defect can be rescued by RNase H overexpression, confirming the causal relationship between R-loops and transcriptional impairment . In cancer research, ChIP assays have revealed that DDX21 binds to the CDK1 promoter in colorectal cancer cells and influences the deposition of H3K4me3 marks, providing mechanistic insight into how DDX21 promotes cancer cell proliferation .

How does DDX21 interact with other proteins to form functional complexes in different cellular contexts?

DDX21 forms context-specific protein complexes that mediate its diverse cellular functions:

Major DDX21 protein complexes:

Methods to study DDX21 protein interactions:

• Co-immunoprecipitation (Co-IP):

  • Lyse cells in NP-40 buffer

  • Precipitate with anti-DDX21 or anti-tag antibodies

  • Identify interacting partners via western blotting or mass spectrometry

  • Confirm direct interactions using recombinant proteins

• Proximity ligation assays:

  • Detect protein-protein interactions in situ

  • Visualize DDX21 interactions within specific subcellular compartments

  • Quantify interaction frequencies under different conditions

• Domain mapping experiments:

  • Use deletion mutants to identify interaction domains

  • Test functional mutants (e.g., helicase-dead K237E, S376L, M4)

  • Determine if interactions are dependent on enzymatic activity

  • Example: DDX21 promotes PRRSV proliferation independently of its enzymatic activities

Research shows that in colorectal cancer, DDX21 directly interacts with WDR5, a core component of histone methyltransferase complexes. This interaction influences the deposition of H3K4me3 marks on the CDK1 promoter, thereby promoting cell cycle progression . In antiviral immunity, DDX21 forms part of the DDX1-DDX21-DHX36 complex that senses viral dsRNA, but caspase-dependent cleavage of DDX21 during infection disrupts this complex and modulates the immune response .

What are the latest findings regarding post-translational modifications of DDX21 and their impact on function?

Post-translational modifications (PTMs) of DDX21 critically regulate its activity, localization, and protein interactions:

Key DDX21 modifications:

• Caspase-dependent cleavage:

  • Occurs at residue D126 during viral infection

  • Mediated by caspase-3/6

  • Promotes translocation from nucleus to cytoplasm

  • Results in negative regulation of IFN-β signaling

  • Suppresses formation of DDX21-containing immune complexes

• Deacetylation by SIRT7:

  • Activates DDX21 helicase activity

  • Enables resolution of R-loops

  • Prevents transcription-associated genomic instability

  • Essential for overcoming R-loop-mediated RNA polymerase stalling

• Phosphorylation:

  • Affects DDX21 helicase activity and interactions

  • Regulates nucleolar/nucleoplasmic distribution

  • Responsive to cellular stresses and metabolic changes

• SUMOylation:

  • Modifies DDX21 localization and function

  • Influences interactions with specific protein partners

  • Potentially regulated during cell cycle progression

Experimental approaches to study PTMs:

• Site-directed mutagenesis:

  • Generate non-cleavable mutants (e.g., D126A for caspase cleavage site)

  • Create phospho-mimetic or phospho-deficient mutants

  • Assess functional consequences through rescue experiments

• Mass spectrometry:

  • Identify specific modification sites

  • Quantify modification changes under different conditions

  • Map modification patterns across the protein

• PTM-specific antibodies:

  • Detect modified forms of DDX21

  • Monitor dynamic changes in modification status

  • Determine subcellular localization of modified DDX21

Recent research has demonstrated that caspase-dependent cleavage of DDX21 at D126 occurs following virus infection and treatment with RNA/DNA ligands, especially for RNA viruses. This cleavage event promotes the translocation of DDX21 from the nucleus to the cytoplasm, where the cleaved form negatively regulates the IFN-β signaling pathway by suppressing the formation of the DDX1-DDX21-DHX36 complex. This mechanism helps maintain immune balance during viral infection .

What are common issues when using DDX21 antibodies and how can they be resolved?

Researchers may encounter several challenges when working with DDX21 antibodies:

Common challenges and solutions:

• Background signal issues:

  • Problem: High background in immunofluorescence or immunohistochemistry

  • Solution: Increase blocking time (2% BSA or 10% serum for 1-2 hours), optimize antibody dilution (typically 1:50-1:500 for IHC, 1:50-1:500 for IF), and include additional washing steps

• Cross-reactivity concerns:

  • Problem: Antibody detects proteins other than DDX21

  • Solution: Validate specificity using DDX21 knockdown controls, CRISPR knockout cells, or blocking peptides. Compare results with multiple antibodies targeting different DDX21 epitopes

• Subcellular localization discrepancies:

  • Problem: Inconsistent localization patterns between experiments

  • Solution: Standardize fixation methods (4% PFA for 20 min), optimize permeabilization (0.2% Triton X-100 for 20 min), and include nucleolar markers (nucleolin, fibrillarin) for co-localization studies

• Detection sensitivity limitations:

  • Problem: Weak signal in western blotting

  • Solution: Use optimized lysis buffers (NP-40 based), increase protein loading, extend transfer time, and optimize antibody concentration (typically 1:500-1:50000 for WB)

• Epitope masking due to protein interactions:

  • Problem: Reduced detection in specific cellular contexts

  • Solution: Use multiple antibodies targeting different DDX21 domains, try different extraction methods, and consider native vs. denaturing conditions depending on application

When selecting DDX21 antibodies, researchers should consider the specific application, target species, and whether post-translational modifications or specific protein complexes are being studied. Validation using multiple approaches, particularly including knockdown or knockout controls, is essential for ensuring specificity and reproducibility.

How can researchers distinguish between different functional states of DDX21?

Distinguishing between different functional states of DDX21 requires specialized approaches:

Methodological strategies:

• Analysis of subcellular localization:

  • Nucleolar DDX21: Associated with rRNA processing and ribosome biogenesis

  • Nucleoplasmic DDX21: Involved in RNA polymerase II regulation and 7SK binding

  • Cytoplasmic DDX21: Often associated with innate immune sensing or cleaved form

  • Use confocal microscopy with specific subcellular markers for precise localization

• Detection of specific protein complexes:

  • Use co-immunoprecipitation followed by western blotting for known partners

  • Apply proximity ligation assays to visualize specific interactions in situ

  • Employ glycerol gradient fractionation to separate distinct DDX21-containing complexes

• Identification of post-translationally modified forms:

  • Use antibodies specific for cleaved DDX21 (if available)

  • Apply phospho-specific antibodies to detect phosphorylated states

  • Employ 2D gel electrophoresis to separate differently modified forms

  • For caspase-cleaved DDX21, look for ~75 kDa fragment vs. full-length 87 kDa protein

• Assessment of enzymatic activity:

  • Measure helicase activity using RNA unwinding assays

  • Evaluate RNA folding capacity through secondary structure formation assays

  • Compare activity of wild-type vs. mutant forms (K237E, S376L, M4)

• RNA-binding analysis:

  • Perform RNA immunoprecipitation (RIP) to identify bound RNAs (rRNAs, snoRNAs, 7SK)

  • Use CLIP-seq to map RNA binding sites genome-wide

  • Compare RNA binding profiles in different cellular contexts

Research has shown that DDX21 undergoes dramatic relocalization during viral infection, moving from the nucleolus to the nucleoplasm during HCMV infection or from the nucleus to the cytoplasm during PRRSV infection . Additionally, caspase-cleaved DDX21 shows distinct functional properties compared to the full-length protein, particularly in regulating innate immune responses .

How can researchers integrate DDX21 studies with broader -omics approaches?

Integrating DDX21 research with -omics approaches provides comprehensive insights into its functions:

Multi-omics strategies:

• Transcriptomic integration:

  • Perform RNA-seq after DDX21 knockdown or overexpression

  • Identify directly and indirectly regulated genes

  • Conduct pathway enrichment analysis on differentially expressed genes

  • Example: Gene Ontology analysis of top 100 genes co-expressed with DDX21 reveals enrichment in RNA processing and ribosome biogenesis pathways

• Proteomic approaches:

  • Use IP-MS (immunoprecipitation coupled with mass spectrometry) to identify DDX21 interactome

  • Compare interactomes across different cell types or conditions

  • Apply quantitative proteomics to measure proteome-wide changes upon DDX21 manipulation

  • Research shows DDX21 interactions with factors involved in RNA processing, transcription, and chromatin modification

• Chromatin profiling:

  • Perform ChIP-seq to map genome-wide DDX21 binding sites

  • Integrate with histone modification data (H3K4me3, H3K27ac)

  • Correlate with transcription factor binding and chromatin accessibility

  • Example: DDX21 binding regions on CDK1 promoter correlate with H3K4me3 enrichment

• RNA-protein interaction mapping:

  • Apply CLIP-seq or PAR-CLIP to identify DDX21-bound RNAs

  • Determine RNA binding motifs and structural preferences

  • Correlate with functional outcomes for bound transcripts

• Integrative bioinformatic analysis:

  • Use tools like GEPIA2 and STRING to analyze co-expression networks

  • Identify key correlation patterns with genes like RPL5, RPL30, ZC3H18, NCL, FTSJ3, and STAU1

  • Apply BioGRID data to map protein-protein interaction networks

  • Pan-cancer analysis through TCGA, GEO, GTEx, CPTAC, and HPA datasets reveals DDX21 as a potential biomarker across multiple cancer types

Recent pan-cancer analysis integrated multiple -omics datasets to identify DDX21 as a potential biomarker for cancer prognosis. This approach combined transcriptomic data from TCGA with protein expression data from the Human Protein Atlas, revealing that DDX21 overexpression correlates with poor survival across multiple cancer types. Co-expression network analysis further identified key DDX21-associated genes involved in RNA processing and cell cycle regulation .