HNRNPDL Antibody

What is HNRNPDL and why is it important in cellular biology?

HNRNPDL (Heterogeneous Nuclear Ribonucleoprotein D-Like) belongs to a class of conserved nuclear RNA-binding proteins (RBPs) that assemble with RNA to form ribonucleoproteins (RNPs). It functions as a transcriptional regulator and participates in the metabolism and biogenesis of mRNA . The protein is particularly significant because:

• It acts as a transcription factor and participates in mRNA metabolism

• It can shuttle between the nucleus and cytoplasm

• It binds to both nuclear and cytoplasmic mRNAs, especially those containing AU-rich elements (AREs)

• Its self-assembly properties contribute to important cellular functions

• Mutations in this protein are associated with limb-girdle muscular dystrophy D3 (LGMD D3)

How many isoforms of HNRNPDL exist and what are their structural differences?

Three isoforms of HNRNPDL are produced by alternative splicing:

What are the molecular characteristics of HNRNPDL protein?

HNRNPDL has several key molecular characteristics:

• Calculated molecular weight: ~46 kDa

• Observed molecular weight in Western blots: 38-40 kDa

• Contains two adjacent RNA binding domains (RBDs)

• Features a glycine-rich C-terminal auxiliary domain

• Forms part of the D-subgroup of hnRNPs along with hnRNP-D and hnRNP-AB

• Has the ability to bind AU-rich elements found in the 3′-UTR of many proto-oncogenes and cytokine mRNAs

• Can form functional amyloid fibrils under certain conditions

How is HNRNPDL linked to muscular dystrophy?

HNRNPDL is associated with limb-girdle muscular dystrophy D3 (LGMD D3) through specific mutations:

• Point mutations in hnRNPDL exon 6 cause autosomal dominant LGMD D3

• Research suggests that LGMD D3 might be a loss-of-function disease associated with impaired fibrillation of HNRNPDL

• Disease-causing mutations in HNRNPDL have been shown to accelerate protein aggregation

• When expressed in Drosophila muscle, these mutant variants become completely insoluble

• The cryo-EM structure of hnRNPDL-2 fibrils reveals that the amyloid core maps to exon 6, which is precisely the location of disease-causing mutations

What is the significance of HNRNPDL as a biomarker in rheumatoid arthritis?

HNRNPDL has emerged as a potential biomarker for rheumatoid arthritis (RA):

• Autoantibodies against both native and citrullinated forms of HNRNPDL have been detected in RA patients

• The citrullinated/native index of autoantibodies against hnRNP-DL (CN DL-Index) has been identified as a new value for an "individual window of treatment success" in early RA

• This index helps detect RF IgM/α-CCP-2 seronegative RA patients (24-46%)

• Patients with a negative CN DL-index tend to be good responders to methotrexate (MTX) treatment (87%)

• High positive CN DL-values are associated with more severe RA, shared epitope and parenchymal changes in the lung

• Native α-hnRNP-DL antibodies show TLR7/9-dependency and are associated with pain

What are the optimal conditions for detecting HNRNPDL using Western blot?

For optimal Western blot detection of HNRNPDL:

When troubleshooting, remember that observed molecular weight (38-40 kDa) may differ from calculated weight (46 kDa) due to post-translational modifications or isoform expression .

What techniques are available for studying HNRNPDL phase separation and aggregation?

Several techniques have been documented for studying HNRNPDL phase separation and aggregation properties:

• Dynamic Light Scattering (DLS):

  • Used to analyze the radii of hydration of HNRNPDL isoforms

  • Can be performed at physiological salt concentration without crowding agents

  • Results correlate with propensity to undergo LLPS

• Microscopy Techniques:

  • Differential interference contrast (DIC) microscopy to observe droplet formation

  • Fluorescence microscopy with tagged proteins to track localization

  • Cryo-electron microscopy (cryo-EM) for high-resolution structural analysis of fibrils

• Cell-based Assays:

  • Expression of fluorescently tagged HNRNPDL isoforms in cells

  • Assessment of intranuclear distribution patterns

  • Analysis of nuclear/cytoplasmic shuttling properties

• In vitro Fibrillation Assays:

  • Protein purification followed by controlled aggregation

  • Thioflavin T fluorescence to monitor amyloid formation

  • Assessment of nucleic acid binding by purified fibrils

How should researchers select and validate HNRNPDL antibodies for specific applications?

When selecting and validating HNRNPDL antibodies for specific applications, consider:

• Application Compatibility:

  • Verify antibody validation for your specific application (WB, IHC, ICC/IF)

  • Different antibodies may perform optimally in different applications

• Isoform Specificity:

  • Determine which HNRNPDL isoform(s) you need to detect

  • Select antibodies raised against epitopes present in your target isoform(s)

  • For hnRNPDL-3 detection, avoid antibodies targeting the C-terminal region

• Validation Methods:

  • Western blot with positive controls (HL-60, HeLa, K-562 cells)

  • Immunohistochemistry with recommended tissues (e.g., human intrahepatic cholangiocarcinoma)

  • Knockdown or knockout validation to confirm specificity

  • Testing in multiple species if cross-reactivity is needed

• Host and Format Considerations:

  • Most commercial HNRNPDL antibodies are rabbit polyclonals

  • Consider conjugated options if needed for multiplexing

  • Storage requirements typically include -20°C with aliquoting recommended

How can researchers differentiate between native and citrullinated forms of HNRNPDL?

Differentiating between native and citrullinated forms of HNRNPDL requires specialized methods:

• ELISA-based Approaches:

  • Develop separate ELISAs using native and citrullinated HNRNPDL as antigens

  • Calculate a CN DL-Index as the difference between citrullinated and native antibody signals

  • This delta value improves diagnostic sensitivity and indicates association to shared epitope

• Protein Expression and Modification:

  • Express recombinant HNRNPDL using systems such as the pET-30 Ek/LIC vector

  • Perform in vitro citrullination using peptidylarginine deiminases (PADs)

  • Verify citrullination by mass spectrometry or using anti-citrulline antibodies

• Structural Citrullination-dependent Epitopes (SCEs):

  • Design assays to detect SCEs of HNRNPDL

  • These epitopes have been detected in 58% of SLE patients despite being α-CCP-2-negative

  • Use paired antibodies that specifically recognize either native or citrullinated forms

• Immunohistochemical Detection:

  • Perform double staining with anti-HNRNPDL and anti-citrulline antibodies

  • HNRNPDL has been shown to be citrullinated in the rheumatoid joint

What is known about the structure of HNRNPDL fibrils and how can this inform research?

The structure of HNRNPDL fibrils has been characterized by cryo-EM, providing valuable insights:

• Structural Features:

  • Full-length hnRNPDL-2 forms amyloid fibrils with stable, non-toxic properties

  • The high-resolution amyloid core consists of a single Gly/Tyr-rich and highly hydrophilic filament

  • The core contains internal water channels

  • RNA binding domains are located as a solenoidal coat around the core

• Functional Implications:

  • HNRNPDL-2 fibrils are stable, non-toxic, and bind nucleic acids

  • Their architecture and activity resemble functional amyloids

  • The fibril core precisely maps to exon 6, which is absent in the soluble hnRNPDL-3 isoform

• Research Applications:

  • The structure provides a basis for studying how alternative splicing controls HNRNPDL assembly

  • It offers insights into how disease-causing mutations might disrupt normal fibril formation

  • Understanding the structure informs approaches to modulate HNRNPDL aggregation

• Disease Relevance:

  • The structural data suggests that LGMD D3 might be a loss-of-function disease associated with impaired fibrillation

  • This contradicts the traditional view of protein aggregation diseases as gain-of-toxic-function disorders

How does alternative splicing regulate HNRNPDL phase separation and what methods can be used to study this phenomenon?

Alternative splicing significantly impacts HNRNPDL phase separation properties, which can be studied through several approaches:

• Comparative Isoform Analysis:

  • Express and purify the three HNRNPDL isoforms

  • DLS analysis shows DL1 forms assemblies with radius >1,000 nm, while DL2 and DL3 display average radii of 4.5 and 3.5 nm respectively

  • DL1 readily undergoes LLPS, DL2 shows reduced propensity, and DL3 completely loses this ability

• Domain Contribution Studies:

  • The absence of the N-terminus Arg-enriched IDR reduces LLPS propensity

  • The absence of both N- and C-terminus IDRs completely abolishes LLPS

  • This demonstrates how alternative splicing precisely controls LLPS by including/excluding specific domains

• Cellular Distribution Analysis:

  • All HNRNPDL isoforms localize to the nucleus but show different distribution patterns

  • DL1 and DL2 are found throughout the nucleoplasm but excluded from nucleoli

  • DL3 shows completely diffuse nuclear distribution, suggesting the C-terminus IDR determines intranuclear compartmentalization

• Role of Key Residues:

  • Arg and Tyr residues in two distant IDRs act as crucial determinants for both LLPS and aggregation

  • This spatial segregation of multivalent interacting residues explains how AS controls high-order assembly formation

  • Mutations affecting these residues can be studied to understand their contribution to phase separation

What are common issues encountered when working with HNRNPDL antibodies and how can they be resolved?

Common issues with HNRNPDL antibodies and their solutions include:

How should researchers design experiments to study the interaction between HNRNPDL and nucleic acids?

When designing experiments to study HNRNPDL-nucleic acid interactions:

• RNA Binding Assays:

  • RNA electrophoretic mobility shift assays (EMSA) using purified HNRNPDL isoforms

  • RNA immunoprecipitation (RIP) to identify bound RNAs in cellular contexts

  • UV crosslinking assays to capture direct interactions

  • Special focus on AU-rich elements (AREs) found in 3′-UTR regions, which are known targets

• Structural Analysis:

  • The RNA binding domains of HNRNPDL-2 fibrils are located as a solenoidal coat around the amyloid core

  • This arrangement allows for nucleic acid binding even in the fibrillar state

  • Cryo-EM can provide insights into the structural basis of these interactions

• Isoform Comparison:

  • All three isoforms contain the RNA recognition motifs (RRMs)

  • Compare binding properties between isoforms to understand how the additional domains influence RNA binding

  • Determine if phase-separated droplets or fibrils retain RNA binding activity

• Disease-Relevant Studies:

  • Examine how disease-causing mutations affect nucleic acid binding

  • Investigate if citrullination alters RNA binding properties

  • Study the potential role of nucleic acids in promoting or inhibiting HNRNPDL aggregation

What considerations are important when using HNRNPDL antibodies for detecting citrullinated forms in autoimmune disease research?

When detecting citrullinated HNRNPDL in autoimmune disease research:

• Epitope Selection:

  • Choose antibodies against regions likely to contain citrullination sites

  • Consider developing paired antibodies that distinctly recognize citrullinated vs. non-citrullinated forms

  • The CN DL-Index approach requires reliable detection of both forms

• Validation in Disease Samples:

  • Use samples from RA patients with known citrullination status

  • Include controls from different autoimmune conditions, especially SLE where SCEs of hnRNP-DL were detected in 58% of patients

  • Verify specificity using competition assays with citrullinated and native peptides

• Technical Considerations:

  • For IHC, perform antigen retrieval with TE buffer pH 9.0 or citrate buffer pH 6.0

  • Consider double staining with anti-citrulline antibodies to confirm modification

  • Process samples quickly to prevent artificial modifications

• Clinical Correlations:

  • Document disease parameters alongside antibody testing

  • The CN DL-Index correlates with disease severity, shared epitope, and lung involvement

  • Native α-hnRNP-DL shows TLR7/9-dependency and associates with pain

  • Stratify patients according to treatment response for meaningful analysis