Journal: The Journal of Experimental Medicine

Article Title: SARS-CoV-2 brainstem encephalitis in human inherited DBR1 deficiency

doi: 10.1084/jem.20231725

Figure Lengend Snippet: A patient with SARS-CoV-2 brainstem encephalitis homozygous for a DBR1 mutation. (A) Family pedigree of index patient 1 (P1) homozygous for the DBR1 I120T/I120T mutation. The segregations of the DBR1 (I120T) and MEFV (M694V, E148Q) variants are indicated. Pathogenic mutations are shown in red for DBR1 and in blue for MEFV . Siblings are labeled from sibling 1 (S1) to S6. (B) Estimation of a potential common haplotype surrounding the DBR1 I120T mutation, as predicted by EstiAge analysis, for P1 and a previously reported patient with HSV-1 BVE. (C) MRI fluid–attenuated inversion recovery images taken on day 2 of encephalitis in P1.

Article Snippet: The membranes were then probed with an anti-human DBR1 antibody (ProteinTech).

Techniques: Mutagenesis, Labeling

Journal: The Journal of Experimental Medicine

Article Title: SARS-CoV-2 brainstem encephalitis in human inherited DBR1 deficiency

doi: 10.1084/jem.20231725

Figure Lengend Snippet: Homozygosity for the I120T DBR1 variant in a child with isolated SCV-2 BVE. (A) VirScan test for antibodies against a wide range of viruses in the serum of P1, his siblings, and their parents. Hierarchically clustered (Pearson) heatmap showing PhIP-Seq antibody enrichment (z-score relative to mock immunoprecipitation [IP]) for each of the 30 viruses detected in at least one member of the family. All values are the means of technical duplicates. (B) Electropherogram showing the DBR1 gDNA sequence surrounding the I120T mutation, in P1 and his older siblings (S1 [born 2000], S2 [born 2002], and S3 [born 2004]). (C) IFN-α, -β, and -γ levels in the plasma of various members of the family and 30 other healthy controls, as measured by SIMOA digital ELISA. Statistical analysis was conducted with Mann–Whitney U tests. ns: not significant.

Article Snippet: The membranes were then probed with an anti-human DBR1 antibody (ProteinTech).

Techniques: Variant Assay, Isolation, Immunoprecipitation, Sequencing, Mutagenesis, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: The Journal of Experimental Medicine

Article Title: SARS-CoV-2 brainstem encephalitis in human inherited DBR1 deficiency

doi: 10.1084/jem.20231725

Figure Lengend Snippet: Intronic RNA lariat levels in patient-derived fibroblasts homozygous for a DBR1 mutation. (A and B) DBR1 mRNA levels (A) and DBR1 protein levels (B) in fibroblasts from two healthy controls, a DBR1 WT/WT sibling (S3), a DBR1 WT/I120T sibling (S1), and a DBR1 I120T/I120T sibling (S2) of P1, P1, and a previously reported DBR1 I120T/I120T patient with HSV-1 brainstem encephalitis. (C) ID1 and DKK1 mRNA and intronic RNA lariat levels, in fibroblasts, as in A and B, as measured by RT-qPCR. Statistical analysis was performed with two-tailed Mann–Whitney U test. ***P < 0.001. (D and E) DBR1 mRNA levels (D) and DBR1 protein levels (E) in fibroblasts from one healthy control and P1 transduced with empty vector, I120T DBR1, or WT DBR1. (F) ID1 and DKK1 intronic RNA lariat levels, in fibroblasts, as in D and E, as measured by RT-qPCR. Statistical analysis was performed with two-tailed Mann–Whitney U test. **P < 0.01. Data from A, C, D, and F are presented as the means ± SEM from three independent experiments, with two biological replicates for each experiment. Data shown in B and E are representative of three independent experiments. Source data are available for this figure: .

Article Snippet: The membranes were then probed with an anti-human DBR1 antibody (ProteinTech).

Techniques: Derivative Assay, Mutagenesis, Quantitative RT-PCR, Two Tailed Test, MANN-WHITNEY, Control, Transduction, Plasmid Preparation

Journal: The Journal of Experimental Medicine

Article Title: SARS-CoV-2 brainstem encephalitis in human inherited DBR1 deficiency

doi: 10.1084/jem.20231725

Figure Lengend Snippet: Intronic RNA lariat levels in hPSC-derived hindbrain neurons homozygous for a DBR1 mutation. (A) Angiotensin-converting enzyme 2 ( ACE2 ) mRNA levels were determined by RT-qPCR in SV-40 transformed fibroblasts (SV40-F) from healthy controls (C1, C2) and P1, A549 lung carcinoma cells with or without ACE2 transduction, and hPSC-derived hindbrain neurons (HB neurons) from a healthy control (H9) and a previously reported patient with the DBR1 mutation (DBR1 I120T/I120T). The data shown are the mean ± SEM from two independent experiments, with two technical replicates for each experiment. The limit of detection (LOD) is set as the median of ACE2 mRNA levels in SV40-F, which does not express ACE2. (B and C) DBR1 mRNA levels (B) and ID1 and DKK1 RNA lariat levels (C) in hindbrain neurons derived from healthy control (H9) and DBR1 I120T/I120T patient hPSCs, as measured by RT-qPCR. The data from B and C are presented as means ± SEM from two independent experiments, with two biological replicates for each experiment. (D) DBR1 mRNA levels and ID1 and DKK1 RNA lariat levels in hindbrain neurons derived from healthy control and DBR1 I120T/I120T patient hPSCs transduced with empty vector, I120T DBR1, or WT DBR1, as measured by RT-qPCR. The data from D are presented as means ± SEM from two independent experiments, with two biological replicates for each experiment. (E) DKK1 lariat RNA levels in hindbrain neurons derived from healthy control and DBR1 I120T/I120T patient hPSCs with or without SARS-CoV-2 infection (MOI 1, 24 hpi). The data shown are the mean ± SEM from three independent experiments, with two technical replicates for each experiment.

Article Snippet: The membranes were then probed with an anti-human DBR1 antibody (ProteinTech).

Techniques: Derivative Assay, Mutagenesis, Quantitative RT-PCR, Transformation Assay, Transduction, Control, Plasmid Preparation, Infection

Journal: The Journal of Experimental Medicine

Article Title: SARS-CoV-2 brainstem encephalitis in human inherited DBR1 deficiency

doi: 10.1084/jem.20231725

Figure Lengend Snippet: Characterization of hPSC-derived hindbrain neurons. (A and B) Abundance of mRNA for the neuronal markers GABAR1, TUBB2B, and SLC17A7 (A), and for the hindbrain neuron–specific markers GBX2, HOXA2, HOXB2, and HOXB4 (B), as assessed by RNAseq, in hPSC-derived hindbrain neurons derived from a healthy control (H9), a DBR1 I120T/I120T patient, an IFNAR1 −/− patient, and a TLR3 −/− patient. SV40-fibroblasts from a healthy control (C1), a DBR1 I120T/I120T patient, an IFNAR1 −/− patient, and a TLR3 −/− patient were included as negative controls for the detection of these neuron-specific markers. Triplicates were studied for each sample in A and B.

Article Snippet: The membranes were then probed with an anti-human DBR1 antibody (ProteinTech).

Techniques: Derivative Assay, Control

Journal: The Journal of Experimental Medicine

Article Title: SARS-CoV-2 brainstem encephalitis in human inherited DBR1 deficiency

doi: 10.1084/jem.20231725

Figure Lengend Snippet: SARS-CoV-2 infection in hPSC-derived hindbrain neurons. (A) Representative immunofluorescence images of hPSC-derived hindbrain neurons infected with SARS-CoV-2 (MOI 0.1) at 72 h post-infection (hpi) for a healthy control (H9), a previously reported patient with the DBR1 mutation (DBR1 I120T/I120T), and patients with complete TLR3 (TLR3 −/− ) or IFNAR1 (IFNAR1 −/− ) deficiency. Cells were stained with antibodies against the SARS-CoV-2 nucleocapsid protein (N, red) and a neuron-specific microtubule-associated protein 2 (MAP2, green). A/T-rich chromosomal DNA was stained with DAPI (blue). Bar: 150 µm. The data shown are representative of three independent experiments. (B and C) Percentage of hindbrain neurons (MAP2 + ) positive for the SARS-CoV-2 N protein, at various time points (hpi), with and without IFN-β pretreatment, for cells infected with SARS-CoV-2 at an MOI of 0.1 (B) or 10 (C). The data points are the means ± SEM from three independent experiments with three technical replicates per experiment. Statistical analysis was conducted with Kruskal–Wallis tests, with Dunn’s test for multiple comparisons. *P < 0.05; **P < 0.01; ***P < 0.001. (D) Scatterplots of the mean log 2 fold-changes in RNAseq-quantified gene induction following stimulation with 100 IU/ml of IFN-β for 8 h, in hPSC-derived hindbrain neurons from a healthy control (H9), a previously reported patient with the DBR1 mutation (DBR1 I120T/I120T), and patients with complete TLR3 (TLR3 −/− ) or IFNAR1 (IFNAR1 −/− ) deficiency. Each point represents a single gene. Genes with an absolute fold-change in expression >2 in response to IFN-β treatment relative to NS samples in the control (Ctrl) group are plotted. (E and F) ID1 and DKK1 intronic RNA lariat levels (E) and SARS-CoV-2 nucleocapsid 2 (SCV-2 N2 ) and RNA-dependent RNA polymerase (SCV-2 RdRp ) mRNA levels (F), in hPSC-derived hindbrain neurons from a healthy control (H9) transduced with DKK1 lariat-expressing lentivirus, as measured by RT-qPCR, after infection with SARS-CoV-2 (MOI 0.1), 2 hpi, 24 hpi and 36 hpi. The data shown are the mean ± SEM from two independent experiments, with two biological replicates for each experiment.

Article Snippet: The membranes were then probed with an anti-human DBR1 antibody (ProteinTech).

Techniques: Infection, Derivative Assay, Immunofluorescence, Control, Mutagenesis, Staining, Expressing, Transduction, Quantitative RT-PCR

Journal: The Journal of Experimental Medicine

Article Title: SARS-CoV-2 brainstem encephalitis in human inherited DBR1 deficiency

doi: 10.1084/jem.20231725

Figure Lengend Snippet: SARS-CoV-2 infection in hPSC-derived hindbrain neurons with and without IFN-β pretreatment. (A) Representative immunofluorescence images of hPSC-derived hindbrain neurons infected with SARS-CoV-2 (MOI 10) at 72 hpi, for a healthy control (H9), a previously reported patient with the DBR1 mutation (DBR1 I120T/I120T), and patients with complete TLR3 (TLR3 −/− ) or IFNAR1 (IFNAR1 −/− ) deficiency. Cells were stained with antibodies against the SARS-CoV-2 nucleocapsid protein (N, red) and a neuron-specific microtubule-associated protein 2 (MAP2, green). A/T-rich chromosomal DNA was stained with DAPI (blue). Bar: 150 µm. Data shown are representative of three independent experiments. (B) Quantification of the SARS-CoV-2 nucleocapsid (N2) (upper panel) and the RNA-dependent RNA polymerase (RdRp) (lower panel) by TaqMan real-time qPCR, at 2, 24, 48, 72, and 96 h after SARS-CoV-2 infection (MOI 1). Data are presented as the mean ± SEM and are representative of two independent experiments with biological triplicates in each experiment. (C) Heatmaps of RNAseq-quantified gene expression (z-score-scaled DESeq2 vst-normalization) in hPSC-derived hindbrain neurons from a healthy control (H9), a previously reported patient with the DBR1 mutation (DBR1 I120T/I120T), an IFNAR1 −/− patient, and a TLR3 −/− H patient, not stimulated (NS) or stimulated with IFN-β for 8 h. Duplicates were studied for each set of conditions and mean gene expression levels were used for subsequent analyses. The heatmap includes genes with a relative fold-change in expression >2 in response to IFN-β treatment relative to NS samples in the control group.

Article Snippet: The membranes were then probed with an anti-human DBR1 antibody (ProteinTech).

Techniques: Infection, Derivative Assay, Immunofluorescence, Control, Mutagenesis, Staining, Gene Expression, Expressing

Journal: Nature communications

Article Title: The debranching enzyme Dbr1 regulates lariat turnover and intron splicing.

doi: 10.1038/s41467-024-48696-1

Figure Lengend Snippet: Fig. 4 | AQR recruits Dbr1 to branchpoints. A For each Dbr1-interacting RNA- binding protein, the change in normalized lariat levels between C22 and 293T samples for introns with and without reported eCLIP binding sites (mean ± SE shown; n = 15087 introns for AQR, 230 introns for HNRNPA1, 2462 introns for HNRNPC, 4322 introns for HNRNPL, 6330 introns for PTBP1, 2576 introns for RBM22 and 280 introns for SRSF7; p-values from two-sided t-test). B For the three RBPs with significant changes in (A), the change in lariat levels between C22 and 293T samples is compared to the location of RBP binding sites relative to the 5’ splice site and branchpoint. C Reciprocal co-IP of Dbr1 and AQR in 293T cells (top;

Article Snippet: MinuteTMTotal Protein Extraction kit (Invent Biotechnologies) was used, and DBR1 knockout werewas confirmed on single colonies #19 (C19) and #22 (C22) by western blot with the following antibodies: rabbit polyclonal anti-Dbr1 (Proteintech, Cat no: 16019-1-AP) at 1:500 and mouse monoclonal anti-GAPDH (Santa Cruz Biotechnology, sc-47724) at 1:200.

Techniques: RNA Binding Assay, Binding Assay, Co-Immunoprecipitation Assay

Journal: Nature Communications

Article Title: The debranching enzyme Dbr1 regulates lariat turnover and intron splicing

doi: 10.1038/s41467-024-48696-1

Figure Lengend Snippet: A Dbr1 protein levels in wild type 293T and DBR1 knockout cells lines (C9, C19, C22) assayed by western blot. B Fluorogenic debranching activity assay of extracts from C9, C19, and C22 cells compared to 293T control lysates ( n = 4 independent experiments for each cell line; sample values shown as colored points; mean ± SE shown in black). C Fluorescent immuno-microscopy of 293T and C22 cells, using DAPI stain and α-Dbr1 polyclonal antibodies. D FISH analysis of Taok2 intron 13 in 293T and C22 cells (left), and quantification of cytoplasmic and nuclear intron foci (right; n = 35 cells for 293 T, 58 cells for C22; center line represents median; lower and upper bounds of the box represent the 25th and 75th percentile, respectively; lower and upper whiskers extend to the smallest or largest value no further than 1.5× the inter-quartile range from the 25th or 75th percentile value, respectively; outliers not shown). Source data are provided as a Source Data file.

Article Snippet: Western blots of the lysates were performed with an anti-Dbr1 polyclonal antibody using standard protocols (Proteintech 16019-1-AP).

Techniques: Knock-Out, Western Blot, Activity Assay, Control, Microscopy, Staining

Journal: Nature Communications

Article Title: The debranching enzyme Dbr1 regulates lariat turnover and intron splicing

doi: 10.1038/s41467-024-48696-1

Figure Lengend Snippet: A The lariat read recovery rate (lariat reads/total mapped reads) in C19, C22 and two 293T control samples. B Fold change between DBR1 KO and wild type samples in the coverage of individual introns that were classified by ShapeShifter as exhibiting lariat accumulation. C Sequence logo of branchsites from lariat reads recovered in 293T and C22 samples from introns with a single recovered branchpoint. D Sequence logo of the top 100 5’ splice sites ranked by lariat read counts in 293T and C22 samples. E Change in lariat levels between C22 and 293T samples for annotated U12 introns as well as U2 introns from the genes containing U12 introns. F Tally of branchpoints reported in previous mapping studies (Pineda and Mercer ) and those found in DBR1 KO cell lines. G Branchpoint nucleotide composition of lariats reads recovered in 293T and C22 samples. H Distribution of the maximum branchpoint functional score in an 11 bp window centered on branchpoints from lariat reads recovered in 293T ( n = 1374 branchpoints) and C22 samples ( n = 15829 branchpoints; p value from two-sided t-test; center line represents median; lower and upper bounds of the box represent the 25th and 75th percentile, respectively; lower and upper whiskers extend to the smallest or largest value no further than 1.5x the inter-quartile range from the 25th or 75th percentile value, respectively; outliers not shown). Source data are provided as a Source Data file.

Article Snippet: Western blots of the lysates were performed with an anti-Dbr1 polyclonal antibody using standard protocols (Proteintech 16019-1-AP).

Techniques: Control, Sequencing, Functional Assay

Journal: Nature Communications

Article Title: The debranching enzyme Dbr1 regulates lariat turnover and intron splicing

doi: 10.1038/s41467-024-48696-1

Figure Lengend Snippet: A Dbr1-FLAG and binding partners were isolated through co-immunoprecipitation with anti-FLAG magnetic beads. The eluate shows a clear band for Dbr1 on a Coomassie-stained gel ( n = 3 independent replicates for both non-transfected control cells and Dbr1-FLAG transfected cells). B Significant interactions between Dbr1 and spliceosome factors Prp8, Prp19, and Cwc2 (red) were detected, and these factors are adjacent to the lariat (green) in cryo-EM structures of the C and intron lariat spliceosome (ILS) complexes. After transition to the ILS complex, the lariat becomes accessible to Dbr1. C The count of human introns with an eCLIP binding site from ENCODE for Dbr1 co-IP partners that are RNA-binding proteins. D Immunofluorescence using anti-AQR and anti-HA antibodies in U2OS cells shows Dbr1 and AQR co-localize to nuclear speckles ( n = 3 independent replicates for each treatment). Source data are provided as a Source Data file.

Article Snippet: Western blots of the lysates were performed with an anti-Dbr1 polyclonal antibody using standard protocols (Proteintech 16019-1-AP).

Techniques: Binding Assay, Isolation, Immunoprecipitation, Magnetic Beads, Staining, Transfection, Control, Cryo-EM Sample Prep, Co-Immunoprecipitation Assay, RNA Binding Assay, Immunofluorescence

Journal: Nature Communications

Article Title: The debranching enzyme Dbr1 regulates lariat turnover and intron splicing

doi: 10.1038/s41467-024-48696-1

Figure Lengend Snippet: A For each Dbr1-interacting RNA-binding protein, the change in normalized lariat levels between C22 and 293T samples for introns with and without reported eCLIP binding sites (mean ± SE shown; n = 15087 introns for AQR, 230 introns for HNRNPA1, 2462 introns for HNRNPC, 4322 introns for HNRNPL, 6330 introns for PTBP1, 2576 introns for RBM22 and 280 introns for SRSF7; p -values from two-sided t-test). B For the three RBPs with significant changes in ( A ), the change in lariat levels between C22 and 293T samples is compared to the location of RBP binding sites relative to the 5’ splice site and branchpoint. C Reciprocal co-IP of Dbr1 and AQR in 293T cells (top; n = 3 independent replicates), and Dbr1 and AQR levels in untreated (UT), non-targeted control (NC) and two AQR -targeted siRNA knockdown samples (KD1 and KD2, bottom; n = 3 independent replicates). D Lariat mapping of RNA-seq data from control and AQR knockdown samples ( n = 3 independent replicates shown in color with mean ± SE in black). E Branchpoint nucleotide composition of lariat reads recovered in control and AQR knockdown samples. F Fold-change in lariat levels between C22 and 293T samples for introns containing the AQR binding motif learned from eCLIP peak sequences (mean ± SE shown; n = 17304 introns for both motif and no motif categories). Source data are provided as a Source Data file.

Article Snippet: Western blots of the lysates were performed with an anti-Dbr1 polyclonal antibody using standard protocols (Proteintech 16019-1-AP).

Techniques: RNA Binding Assay, Binding Assay, Co-Immunoprecipitation Assay, Control, Knockdown, RNA Sequencing

Journal: Nature Communications

Article Title: The debranching enzyme Dbr1 regulates lariat turnover and intron splicing

doi: 10.1038/s41467-024-48696-1

Figure Lengend Snippet: A – D Quantitative (top) and qualitative (bottom) changes due to DBR1 KO in transcripts ( A ), lariats ( B ), miRNAs ( C ) and snoRNAs ( D ). Source data are provided as a Source Data file.

Article Snippet: Western blots of the lysates were performed with an anti-Dbr1 polyclonal antibody using standard protocols (Proteintech 16019-1-AP).

Techniques: