| 2005 |
Yeast Dbr1 is a manganese-dependent RNA debranching enzyme that cleaves the 2'-5' phosphodiester bonds of lariat introns. Alanine scanning mutagenesis identified 13 conserved residues (His13, Asp40, Arg45, Asp49, Tyr68, Tyr69, Asn85, His86, Glu87, His179, Asp180, His231, His233) required for function in vivo; mutation of Asp40, Asn85, His86, His179, His231, or His233 to alanine abolished or greatly diminished debranching activity in vitro. Dbr1 sediments as a monomer and requires manganese as the metal cofactor. |
In vitro debranching assays with natural lariat RNAs and synthetic branched RNAs; alanine-scanning mutagenesis of 28 conserved residues; sedimentation analysis |
Nucleic acids research |
High |
16275784
|
| 2000 |
Human DBR1 (hDBR1) encodes a functional RNA lariat debranching enzyme: recombinant hDBR1 expressed in E. coli showed debranching activity in vitro, and the cDNA complemented both the intron accumulation phenotype of S. cerevisiae dbr1 null mutants and the intron accumulation and slow growth phenotypes of S. pombe dbr1 null mutants. |
In vitro debranching assay with recombinant protein; interspecies complementation of S. cerevisiae and S. pombe dbr1 null mutants |
Nucleic acids research |
High |
10982890
|
| 2014 |
Crystal structures of Dbr1 alone and in complex with synthetic RNA compounds mimicking the lariat branchpoint revealed the molecular basis for 2',5'-phosphodiester recognition and explained why Dbr1 lacks activity toward 3',5'-phosphodiester linkages. Functional data on Dbr1 variants confirmed structure-function relationships. |
X-ray crystallography of apo-Dbr1 and Dbr1–branched RNA complexes; functional assays on Dbr1 variants |
Nucleic acids research |
High |
25123664
|
| 2016 |
Entamoeba histolytica Dbr1 contains nearly stoichiometric Fe and Zn per polypeptide; Fe partitions primarily to the β-metal pocket and Zn to the α-pocket. Apo-Dbr1 reconstituted with Fe(II)+Zn(II) is active (~3–4 s⁻¹), while Mn(II) or Fe(II) under aerobic conditions yields inactive enzyme. Co-crystal structures of catalytic mutant H91A with 7-mer and 16-mer branched RNAs show a bridging hydroxide positioned for nucleophilic attack of the scissile phosphate. |
X-ray crystallography (Fe-edge anomalous diffraction); fluorogenic bRNA kinetic assay; metal reconstitution of apoenzyme under aerobic and anaerobic conditions; elemental analysis |
Proceedings of the National Academy of Sciences of the United States of America |
High |
27930312
|
| 2016 |
Yeast Dbr1 active site analysis established: (i) Cys11 is an essential active-site residue, extending the Cys11-Xaa-His13 motif as the metal-binding element (atypical within the metallophosphoesterase superfamily); (ii) His86 acts as a general acid catalyst that protonates the O2' leaving group of the 2'-5' phosphodiester; (iii) Dbr1 adheres to a two-metal catalytic mechanism. Dbr1 also exhibits vigorous Mn²⁺-dependent phosphodiesterase activity toward bis-p-nitrophenylphosphate, which does not require His86. |
Alanine-scanning mutagenesis; in vitro phosphodiesterase assay with bis-p-nitrophenylphosphate and branched RNA substrates; structure-activity analysis |
RNA (New York, N.Y.) |
High |
27765821
|
| 2022 |
Saccharomyces cerevisiae Dbr1 contains stoichiometric Fe and Zn; Fe²⁺ is the most effective cofactor for reconstituting activity in apoenzyme (turnover ~9.2 s⁻¹). Treatment of human lymphoblastoid cells with the iron chelator deferoxamine caused a ~2-fold increase in cellular lariat levels, indicating Fe is an important biological cofactor for Dbr1. |
Elemental analysis (ICP-MS); apoenzyme reconstitution with Fe²⁺, Zn²⁺, Mn²⁺; fluorogenic bRNA kinetic assay; deferoxamine treatment of human cells with lariat quantification |
RNA (New York, N.Y.) |
High |
35459748
|
| 2022 |
A product-bound crystal structure of E. histolytica Dbr1 co-crystallized with a phosphorothioate-branched RNA (PS-bRNA) showed in-crystal hydrolysis of the phosphorothioate bond, revealing a product-bound active site state. Dbr1 cleaves phosphorothioate linkages ~10,000-fold more slowly than native phosphate linkages. The structure suggests product inhibition may contribute to the kinetic mechanism. |
X-ray crystallography of EhDbr1 with PS-bRNA; kinetic comparison of phosphorothioate vs native substrate cleavage |
Biochemistry |
High |
36484984
|
| 2023 |
Human Dbr1 contains a disordered C-terminal domain (characterized by NMR and sequence analysis) that stabilizes Dbr1 by reducing aggregation but is dispensable for debranching activity. The noncatalytic protein Drn1 and TTDN1 (MPLKIP) directly bind human Dbr1. Addition of TTDN1 to in vitro debranching reactions increases the catalytic efficiency of human Dbr1 19-fold but has no effect on E. histolytica Dbr1, which lacks the disordered C-terminal domain. Dbr1 requires Fe²⁺ for efficient catalysis. The identity of the branchpoint nucleotide affects debranching rates. |
NMR spectroscopy; in vitro binding assays (pulldown); in vitro debranching kinetics with TTDN1; comparative analysis with E. histolytica Dbr1; sequence analysis |
The Journal of biological chemistry |
High |
37507019
|
| 2023 |
MPLKIP (TTDN1) interacts with core splicing factors and the lariat debranching protein DBR1 (identified by mass spectrometry-based interaction proteomics). MPLKIP-deficient primary fibroblasts have reduced steady-state DBR1 protein levels, indicating MPLKIP stabilizes DBR1. |
Mass spectrometry-based interaction proteomics (co-immunoprecipitation); immunoblot of MPLKIP-deficient fibroblasts |
EMBO molecular medicine |
Medium |
37800682
|
| 2024 |
DBR1 is the sole debranching enzyme in human cells (demonstrated by knockout). The predominantly nuclear Dbr1 preferentially debranches substrates containing canonical U2 binding motifs and shows specificity for particular 5' splice site sequences. Dbr1 is recruited to the branchpoint through the intron-binding protein AQR (identified by co-immunoprecipitation mass spectrometry). DBR1 depletion causes a 20-fold increase in lariats, increases exon skipping, and causes spliceosomal components to remain associated with lariats for longer, demonstrating a role for Dbr1 in spliceosome recycling. |
DBR1 knockout cell line generation; co-immunoprecipitation mass spectrometry; ADAR-fusion lariat timestamping; transcriptomic analysis; spliceosome association assays |
Nature communications |
High |
38816363
|
| 2024 |
Accumulation of RNA lariats in human DBR1-deficient cells interferes with stress granule (SG) assembly by promoting proteasome-mediated degradation of G3BP1 and G3BP2. Impaired SG assembly reduces PKR recruitment and activation, impairing antiviral immunity against HSV-1. Genetic ablation of PKR abolished the antiviral effect of DBR1 in vitro. Dbr1Y17H/Y17H mice are susceptible to viral infections and show decreased G3BP1/2 expression and reduced PKR phosphorylation in cells and brain. |
DBR1-deficient human cell lines; PKR knockout epistasis; Dbr1Y17H/Y17H mouse model; proteasome inhibition; immunoblot for G3BP1/2 and p-PKR; antiviral infection assays |
The Journal of experimental medicine |
High |
39636299
|
| 2024 |
DBR1 I120T/I120T homozygous fibroblasts from patients with inherited DBR1 deficiency have low DBR1 protein levels and high RNA lariat accumulation. Exogenous WT DBR1 expression in patient-derived DBR1 I120T/I120T fibroblasts and hPSC-derived hindbrain neurons rescued the RNA lariat accumulation phenotype. Expression of exogenous RNA lariats (mimicking DBR1 deficiency) increased susceptibility of WT hindbrain neurons to SARS-CoV-2 infection, indicating that lariat accumulation per se impairs antiviral immunity. |
Patient fibroblast analysis; lentiviral rescue with WT DBR1; hPSC-derived hindbrain neuron differentiation; SARS-CoV-2 infection assay; exogenous lariat transfection |
The Journal of experimental medicine |
High |
39023559
|
| 2017 |
DBR1 knockdown in HIV-infected cells inhibits formation of intermediate and full-length HIV-1 cDNA without affecting minus-strand strong-stop cDNA. This inhibition occurs in the nucleus or perinuclear region: when nuclear import of the HIV-1 reverse transcription complex is blocked by a truncated CPSF6, reverse transcription is completed in the cytoplasm in a DBR1-independent manner. In vitro incubation with yeast or human DBR1 (but not catalytically inactive DBR1(N85A)) resolved a lariat-like structure at the 5' end of HIV-1 RNA detected in infected DBR1-knockdown cells. HIV-1 RNA from DBR1-knockdown cells was resistant to RNase R (which degrades linear but not circular/lariat RNAs), supporting formation of a lariat-like structure. |
shRNA knockdown; cell fractionation; CPSF6 truncation for nuclear import block; 5' RACE; RNase R treatment; in vitro DBR1 treatment with catalytic mutant control |
Journal of virology |
Medium |
24672043 28931690
|
| 2005 |
siRNA-mediated knockdown of human DBR1 expression (reducing mRNA by ~80%) led to decreases in HIV-1 cDNA and protein production; this effect was reversed by cotransfection of a DBR1 cDNA, confirming specificity of the DBR1 requirement for HIV-1 replication. |
siRNA knockdown; DBR1 cDNA rescue; HIV-1 cDNA quantification; viral protein detection |
Retrovirology |
Medium |
16232320
|
| 2017 |
hDBR1 modulates snRNP recycling to affect alternative RNA splicing. Insufficient hDBR1 leads to higher rates of exon skipping confirmed by transcriptomic sequencing. Wild-type p53 and HIF-1 co-regulate hDBR1 expression. Higher hDBR1 expression only affected exon-skipping activity in malignant (not normal) cells. |
siRNA knockdown; transcriptomic sequencing; metabolite profiling; in vivo tumor models; reporter assays for splicing |
Oncogene |
Medium |
28504715
|
| 2026 |
hDBR1 physically associates with spliceosome components, intron-turnover factors (including AQR), and RNA quality-control proteins (UPF1, XRN2, DHX29) as identified by immunopurification coupled to mass spectrometry. RNase A treatment identified an RNA-dependent subnetwork enriched for stress-granule proteins and hnRNPs. Phosphoproteomic profiling identified multiple hDBR1 phosphorylation sites, including four residues preferentially detected after RNase treatment. |
Immunopurification coupled to mass spectrometry (gel-based and on-bead workflows); RNase A treatment; phosphoproteomic profiling |
Cell stress & chaperones |
Medium |
41999910
|
| 2024 |
In DBR1-null cells, lariats accumulate in the cytosol and dsRNA (formed by inverted Alu element hairpins within introns) becomes enriched. Chronic exposure to accumulated lariats attenuates dsRNA sensors MDA5, RIG-I, RNase L, and PKR, reducing the antiviral response. Lariats are transiently elevated during infection (HSV-1, influenza, KSHV). |
DBR1 knockout cells; dsRNA immunofluorescence and quantification; antiviral pathway activation assays; infection experiments with HSV-1, influenza, KSHV |
bioRxivpreprint |
Medium |
bio_10.1101_2024.12.07.627371
|