DBR1 Human

What is DBR1 and what is its primary function in human cells?

DBR1 (RNA lariat debranching enzyme 1) is the sole enzyme in human cells capable of selectively hydrolyzing the 2′-5′ phosphodiester linkage found in RNA lariats . These lariat structures are formed during pre-mRNA splicing when introns are excised by the spliceosome, creating a characteristic branch configuration. The debranching activity of DBR1 is essential for the proper turnover of these excised introns, allowing for their subsequent degradation and recycling of spliceosomal components . Without this enzymatic activity, lariat RNAs accumulate in cells, which can interfere with normal cellular processes including antiviral responses .

What is the structural composition of human DBR1?

Human DBR1 consists of approximately 544 amino acid residues with two distinct domains: an N-terminal metalloprotein phosphoesterase (MPE) domain spanning approximately the first 300 amino acids, and a C-terminal domain (residues ~350-544) that lacks homology to any annotated functional domain . The MPE domain contains the catalytic site responsible for debranching activity and aligns with many other MPE enzymes. Notably, the C-terminal domain is intrinsically disordered, as confirmed by both PONDR prediction software and AlphaFold modeling . This disordered region helps stabilize DBR1 by reducing protein aggregation but is not essential for the core debranching activity .

How does DBR1 deficiency affect cellular processes?

DBR1 deficiency causes multiple downstream effects:

• Lariat accumulation: A 20-fold increase in RNA lariat structures is observed in DBR1 knockout cells .

• Disrupted stress granule (SG) assembly: The accumulation of RNA lariats interferes with the formation of stress granules by promoting proteasomal degradation of key SG components, particularly G3BP1 and G3BP2 .

• Impaired antiviral immunity: Defective SG assembly leads to impaired PKR recruitment, activation, and subsequent antiviral activity against viruses including HSV-1 .

• Increased exon skipping: DBR1 depletion affects alternative splicing patterns, particularly increasing exon skipping events .

• Delayed spliceosome recycling: In the absence of DBR1, spliceosomal components remain associated with lariats for longer periods, potentially reducing the availability of splicing factors for subsequent splicing events .

What methods are most effective for studying DBR1 enzymatic activity in vitro?

To effectively study DBR1 debranching activity in vitro, researchers should consider the following methodological approach:

Protein expression and purification:

• Express human DBR1 in prokaryotic systems like E. coli with appropriate purification tags

• Consider expressing truncated versions (particularly removing the disordered C-terminal domain) to improve stability

• Ensure appropriate metal cofactors, particularly Fe²⁺, which has been demonstrated as required for efficient catalysis

Substrate preparation:

• Synthesize branched RNA substrates with 2'-5' phosphodiester bonds

• Design substrates with canonical U2 binding motifs as DBR1 shows preference for these sequences

• Include variations in 5' splice site sequences to account for DBR1's sequence specificity

Activity assays:

• Monitor debranching through gel electrophoresis to visualize the linearization of branched substrates

• Measure reaction kinetics under various conditions (pH, temperature, ionic strength)

• Include control proteins (such as purified IgG) to ensure specificity of results

Enhancing activity:

• Co-express or add purified interacting partners such as TTDN1, which can increase catalytic efficiency by 19-fold

• Include Drn1 (Cwf19-L1), which forms a complex with DBR1 and may enhance turnover of branched RNA

How can researchers generate viable DBR1 knockout cell lines?

Generation of DBR1 knockout cell lines has been critical for understanding DBR1 function. A methodological approach includes:

• Selection of appropriate cell line: HEK293T cells have been successfully used to generate viable DBR1 knockout lines .

• CRISPR-Cas9 gene editing:

  • Design gRNAs targeting exons encoding catalytic residues

  • Transfect cells with CRISPR-Cas9 components

  • Screen clones for DBR1 knockout using genomic PCR, Western blotting, and debranching activity assays

• Verification approaches:

  • Confirm absence of DBR1 protein by mass spectrometry

  • Verify loss of debranching activity using branched RNA substrates

  • Demonstrate lariat accumulation through lariat sequencing

• Rescue experiments:

  • Re-express DBR1 through transfection to confirm phenotypes are directly caused by DBR1 loss

  • Include control transfections (e.g., with RFP expression plasmids) to control for transfection artifacts

• Complementary approaches:

  • Generate cell lines with catalytically inactive DBR1 mutations

  • Consider conditional knockout systems if constitutive knockout affects cell viability

What techniques can be used to detect and quantify RNA lariat accumulation?

To effectively study lariat accumulation in DBR1-deficient cells, researchers can employ several complementary techniques:

Lariat sequencing:

• Perform specialized RNA-seq focusing on branch points

• Use computational pipelines designed to detect and quantify branch point reads

• Compare abundance profiles between wild-type and DBR1-deficient cells

RT-PCR assays:

• Design primers that span the branch point to specifically amplify lariat structures

• Develop quantitative RT-PCR protocols to measure relative lariat abundance

Single-cell FISH (Fluorescence In Situ Hybridization):

• Design probes specific to intron sequences

• Use this approach to visualize lariat distribution and abundance within individual cells

Reporter systems:

• Develop splicing reporters that generate easily detectable lariats

• Measure accumulation through fluorescence or other readouts

ADAR fusion timestamping:

• Utilize ADAR (adenosine deaminase acting on RNA) fusions to "timestamp" lariats

• This approach can determine how long spliceosomal components remain associated with lariats

What is the relationship between DBR1 and stress granule assembly?

The relationship between DBR1 and stress granule (SG) assembly represents a critical link between RNA processing and antiviral immunity:

Mechanism of interference:

• In DBR1-deficient cells, accumulated RNA lariats interfere with stress granule assembly through promoting proteasome-mediated degradation of key SG components, particularly G3BP1 and G3BP2 .

• This interference creates a deficit in these essential SG proteins, preventing proper SG formation in response to cellular stress.

Experimental approaches to study this relationship:

• Immunofluorescence microscopy: Visualize SG formation using antibodies against G3BP1/2 under stress conditions in wild-type versus DBR1-deficient cells.

• Protein stability assays:

  • Measure half-life of G3BP1/2 using cycloheximide chase experiments

  • Test if proteasome inhibitors (e.g., MG132) rescue G3BP1/2 protein levels

• Rescue experiments:

  • Determine if overexpression of G3BP1/2 can restore SG assembly in DBR1-deficient cells

  • Test if G3BP1/2 stabilization affects antiviral responses

• Mechanistic investigations:

  • Analyze direct vs. indirect interactions between lariats and the proteasome machinery

  • Identify molecular adapters that may link lariat accumulation to enhanced G3BP1/2 degradation

How does DBR1 contribute to antiviral immunity?

DBR1's role in antiviral immunity operates through a molecular pathway involving stress granules and PKR activation:

DBR1-PKR-antiviral pathway:

• Normal DBR1 activity prevents lariat accumulation

• This allows proper stress granule assembly with sufficient G3BP1/2

• Stress granules recruit PKR (protein kinase R)

• PKR becomes activated and initiates antiviral responses

• This pathway is effective against viruses including HSV-1

Experimental evidence and approaches:

• DBR1-deficient patients exhibit susceptibility to viral encephalitis

• Dbr1^Y17H/Y17H mice show similar viral susceptibility patterns

• Brain samples from these mice display decreased G3BP1/2 expression and reduced PKR phosphorylation

• Genetic ablation of PKR abolishes the antiviral effect of DBR1 in vitro

Research methodologies to investigate this pathway:

• Viral infection models:

  • Challenge DBR1-deficient cells with various viruses and measure viral replication

  • Test antiviral drug efficacy in DBR1-deficient vs. normal cells

• PKR activation assays:

  • Measure PKR phosphorylation levels via Western blotting

  • Analyze downstream signaling events in the PKR pathway

• Compound screening:

  • Identify molecules that can bypass the DBR1 requirement for PKR activation

  • Develop potential therapeutic approaches for DBR1-deficient patients

How do protein interactions modulate DBR1 function?

DBR1 interacts with several proteins that modulate its activity and potentially its localization:

Key interacting partners and their effects:

Methodological approaches to study these interactions:

• Protein-protein interaction assays:

  • Co-immunoprecipitation followed by mass spectrometry (Co-IP-MS)

  • Yeast two-hybrid screening

  • Proximity labeling approaches (BioID, APEX)

• Functional assays:

  • In vitro debranching assays with and without interacting partners

  • Mutagenesis of interaction domains

  • Domain swapping experiments

• Structural studies:

  • Cryo-EM of DBR1-interactor complexes

  • NMR studies of the disordered C-terminal domain with binding partners

What is the role of DBR1 in alternative splicing regulation?

DBR1 influences alternative splicing through its impact on spliceosome recycling:

Mechanism of action:

• DBR1 depletion leads to increased exon skipping events

• In the absence of DBR1, spliceosomal components remain associated with lariats for longer periods

• As splicing occurs co-transcriptionally, delayed spliceosome recycling can affect the splicing of downstream exons

• This increases the likelihood that downstream exons will be available for exon skipping events

Experimental approaches:

• RNA-seq analysis:

  • Compare splicing patterns between wild-type and DBR1-deficient cells

  • Quantify different types of alternative splicing events (exon skipping, intron retention, etc.)

• Spliceosome recycling assays:

  • Use ADAR fusion proteins to "timestamp" how long spliceosomal components remain associated with lariats

  • Develop pulse-chase experiments to track spliceosome component availability

• Co-transcriptional splicing analysis:

  • Utilize nascent RNA sequencing methods (e.g., NET-seq, TT-seq)

  • Analyze the relationship between transcription rate and splicing outcomes

• Minigene reporter assays:

  • Design reporters containing alternative exons

  • Evaluate how DBR1 status affects splicing choices

How can understanding DBR1 function contribute to therapeutic approaches?

The involvement of DBR1 in viral immunity and RNA processing suggests several therapeutic applications:

Potential therapeutic approaches:

• Viral encephalitis treatment:

  • Developing compounds that can restore PKR activation in DBR1-deficient cells

  • Targeting the proteasomal degradation of G3BP1/2 to maintain stress granule formation

• Cancer applications:

  • DBR1 dysfunction has been implicated in cancer

  • Investigating how modulation of DBR1 activity affects cancer cell proliferation and survival

• Neurological disorders:

  • Exploring the connection between DBR1 activity and ALS, which has been suggested in previous research

  • Investigating how splicing alterations due to DBR1 deficiency might contribute to neurological phenotypes

Research methodologies:

• Drug screening:

  • High-throughput screens for compounds that bypass DBR1 requirements

  • Structure-based drug design targeting DBR1-interacting proteins

• Precision medicine approaches:

  • Developing diagnostic tools to identify patients with DBR1 deficiency

  • Creating personalized treatment strategies based on DBR1 status

What techniques best characterize DBR1 substrate specificity?

Understanding DBR1's substrate preferences is critical for fully elucidating its biological roles:

Current knowledge:

• DBR1 preferentially debranches substrates with canonical U2 binding motifs

• The enzyme also exhibits specificity for particular 5' splice site sequences

• This specificity suggests that branchsites discovered through sequencing may not necessarily represent those favored by the spliceosome

Methodological approaches:

• In vitro selection (SELEX):

  • Generate libraries of branched RNAs with randomized sequences around the branch point

  • Select for efficiently debranched substrates

  • Sequence to determine preferred motifs

• Systematic mutagenesis:

  • Create a panel of branched RNA substrates with defined sequence variations

  • Measure debranching efficiency for each substrate

  • Develop position-specific scoring matrices for DBR1 preferences

• Structural biology:

  • Obtain structures of DBR1 bound to different substrate RNAs

  • Identify key interactions that determine specificity

  • Use this information to predict DBR1 activity on novel substrates

• Computational approaches:

  • Develop machine learning models trained on experimental data

  • Predict DBR1 activity on any given branched RNA sequence

  • Integrate with transcriptome-wide analyses