Journal: Cell reports

Article Title: Splicing quality control mediated by DHX15 and its G-patch activator SUGP1

doi: 10.1016/j.celrep.2023.113223

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: DNA repair template plasmids containing DHX15-FKBP F36V -2xHA_P2A_BFP, DHX15-FKBP F36V -2xHA_P2A_HygR, and DHX38-FKBP F36V -2xHA_P2A_BFP were synthesized with 800bp dsDNA homology arms by Genewiz and assembled using the NEBuilder HiFi DNA assembly cloning kit (NEB E5520S).

Techniques: Recombinant, Protease Inhibitor, Magnetic Beads, Clone Assay, Isolation, CRISPR, Software, Imaging

Journal: Cell reports

Article Title: Splicing quality control mediated by DHX15 and its G-patch activator SUGP1

doi: 10.1016/j.celrep.2023.113223

Figure Lengend Snippet: (A) Diagram of DHX15’s canonical role in disassembling of intron-lariat spliceosome (ILS) at the end of the splicing cycle and its proposed role in disassembling aberrant splicing intermediates during splicing QC. (B) Schematic of HEK.dDHX15 cell line construction and total and nascent chromatin-associated RNA-seq experiments following rapid dTAG13-induced proteolysis of endogenously FKBPF36V degron-tagged DHX15. (C) Immunoblots of total cell lysates from parental HEK293T.A2 cells and two monoclonal HEK.dDHX15 cell lines were treated with DMSO versus dTAG13 at 100 nM for 2 h. (D) Immunoblots of cytoplasm, nucleoplasm, and chromatin fractions, collected upon the preparation of chromatin-associated nascent RNA. β-actin, cytoplasmic marker; SC35, nucleoplasm and nuclear speckle marker; Histon H3, chromatin-associated protein marker. (E) Illustration of how SI is computed by taking the ratio between spliced exon-exon junction reads and normalized total counts of spliced plus unspliced junction-spanning (exon-intron and intron-exon junction) reads. (F) Example chRNA and totalRNA-seq read coverage of the indicated RBM5 intron in control (DMSO) versus dTAG13-treated HEK.dDHX15 cells. ΔSI is shown. (G) Volcano plots of introns with altered splicing efficiency across six biological replicates upon dTAG13-induced DHX15 depletion. Red/blue, introns exhibiting significant (false discovery rate [FDR] ≤ 0.05) increases/decreases of SI ≥ 0.05; gray, introns exhibiting insignificant or unaltered changes in SI. (H) Scatterplot between chromatin-associated splicing efficiency index (SI.ch) and post-transcriptional splicing efficiency index (SI.postTx) across six biological replicates. (I) Scatterplot (left) and empirical cumulative distribution function (eCDF) plot (right) of DHX15-altered introns and their splicing efficiency changes between steady-state and chromatin-associated nascent state (SI.postTx), across six biological replicates. Magenta/navy (up/down), introns exhibiting significant (FDR ≤ 0.05) increases/decreases of SI ≥ 0.05 upon dTAG13-induced DHX15 depletion in HEK.dDHX15 cells; gray (Unch), introns exhibiting insignificant or unaltered changes in SI. Statistical significance is calculated by Welch’s t test, indicated by asterisks ****p < 0.0001), unless otherwise indicated. See also Figure S1.

Article Snippet: DNA repair template plasmids containing DHX15-FKBP F36V -2xHA_P2A_BFP, DHX15-FKBP F36V -2xHA_P2A_HygR, and DHX38-FKBP F36V -2xHA_P2A_BFP were synthesized with 800bp dsDNA homology arms by Genewiz and assembled using the NEBuilder HiFi DNA assembly cloning kit (NEB E5520S).

Techniques: RNA Sequencing Assay, Western Blot, Marker

Journal: Cell reports

Article Title: Splicing quality control mediated by DHX15 and its G-patch activator SUGP1

doi: 10.1016/j.celrep.2023.113223

Figure Lengend Snippet: (A) Diagram of splicing QC via the rejection route: DHX15 dissembles splicing intermediates rejected by exon-joining helicase DHX38 to facilitate spliceosomal components recycling and intron-lariat degradation. (B) Immunoblots of total cell lysates from parental HEK293T.A2 cells and two monoclonal HEK.dDHX38 cell lines were treated with DMSO versus dTAG13 at 100 nM for 2 h. (C) Volcano plots of introns with altered splicing efficiency averaged across six biological replicates upon dTAG13-induced DHX38 depletion. Red/blue, introns exhibiting significant (FDR ≤ 0.05) increases/decreases of SI ≥ 0.05; gray, introns exhibiting insignificant or unaltered changes in SI. Left, nascent RNA-seq; right, total RNA-seq. (D) Nascent RNA-seq read coverage of the indicated PRPF31 intron in control (DMSO) versus dTAG13-treated HEK.dDHX38 and HEK.dDHX15 cells. ΔSI is shown by taking the SI difference between dTAG13-treated and control cells. (E) Nascent RNA-seq read coverage of SUPT16H’s two introns (as indicated) with different sensitivity to dTAG13-mediated DHX38 versus DHX15 depletions. (F and G) (F) Venn diagram of shared introns (left) with decreased splicing efficiency, and shared genes (right) with introns that exhibit decreased splicing efficiency upon DHX15 and DHX38 depletion, across six biological replicates each. The size of the intersection and odds ratios (ORs) are shown. Statistical significance of the intersection is calculated by Hypergeometric test in R (****p < 0.0001) (G) Gene Ontology (GO) enrichment of the shared and unique substrate genes between DHX38 and DHX15. Enriched Biological Process (BP) GO terms are shown. Adjusted p value was calculAdjusted p value was calculated by Benjamini-Hochberg method. Seeated by Benjamini-Hochberg method. See also Figure S1.

Article Snippet: DNA repair template plasmids containing DHX15-FKBP F36V -2xHA_P2A_BFP, DHX15-FKBP F36V -2xHA_P2A_HygR, and DHX38-FKBP F36V -2xHA_P2A_BFP were synthesized with 800bp dsDNA homology arms by Genewiz and assembled using the NEBuilder HiFi DNA assembly cloning kit (NEB E5520S).

Techniques: Western Blot, RNA Sequencing Assay

Journal: Cell reports

Article Title: Splicing quality control mediated by DHX15 and its G-patch activator SUGP1

doi: 10.1016/j.celrep.2023.113223

Figure Lengend Snippet: (A) eCDF of splice sites strength MaxEntScan scores. Magenta/navy (up/down), introns exhibiting significant (FDR ≤ 0.05) increases/decreases of SI ≥ 0.05 upon dTAG13-induced DHX15 depletion in HEK.dDHX15 cells; gray (Unch), introns exhibiting insignificant or unaltered changes in SI. 5ss, 5′ splice site; 3ss, 3′ splice site. (B) Similar to (A), the distribution of counts of branchpoint site (BPS) per intron. (C) Similar to (B), the distribution of distances between BPS and 3ss for each BPS-3ss pair. Statistical significance in (A)–(C) is calculated by Welch’s t test, indicated by asterisks ****p < 0.0001; ns, not significant) unless otherwise indicated. (D and E) (D) Fraction of proximal versus distal alternative 3ss and 5ss usage and (E) fraction of cryptic splicing status in the down, up, or unchanged intron groups upon dTAG13-induced DHX15 depletion. SJ, splicing junction. Statistical significance in (D)–(E) is calculated by chi-squared test, indicated by asterisks (****p < 0.0001, ***p< 0.001, **p < 0.01) unless otherwise indicated. See also Figure S2.

Article Snippet: DNA repair template plasmids containing DHX15-FKBP F36V -2xHA_P2A_BFP, DHX15-FKBP F36V -2xHA_P2A_HygR, and DHX38-FKBP F36V -2xHA_P2A_BFP were synthesized with 800bp dsDNA homology arms by Genewiz and assembled using the NEBuilder HiFi DNA assembly cloning kit (NEB E5520S).

Techniques:

Journal: Cell reports

Article Title: Splicing quality control mediated by DHX15 and its G-patch activator SUGP1

doi: 10.1016/j.celrep.2023.113223

Figure Lengend Snippet: (A) Diagram of the 22 human G-patch domain-containing proteins annotated in ProRule (PRU00092), excluding the 13 retroviral genes. G-patch factors are grouped together based on their subcellular localizations annotated in the Human Protein Atlas.38 Spliceosomal DHX interaction partners (colored dots) are based on BioGRID annotations.37 (B) The number of introns with altered splicing efficiency upon knockdown of corresponding GPs. Magenta/navy (up/down), counts of introns exhibiting significant changes in splicing (|ΔSI| ≥ 0.05, FDR ≤ 0.05) upon corresponding GP factor knockdown. (C) Intersection UpSet plot of altered introns (|ΔSI| ≥ 0.05, FDR ≤ 0.05) upon depletion of DHX15 and KD of GPs SUGP1, GPKOW, TFIP11, RBM17, and RBM10. (D) Heatmap and hierarchical clustering of 1,167 introns with altered splicing efficiency (|ΔSI| ≥ 0.05, FDR ≤ 0.05) upon depletion of DHX15 that are also altered in any one of the five GP knockdown experiments. Clustering distance = 1 – Pearson’s correlation. Cryptic_SJ, cryptic intron status annotated based on their splicing junctions. (E) Heatmap of Pearson correlation matrix constructed on the introns as in (D). See also Figure S3.

Article Snippet: DNA repair template plasmids containing DHX15-FKBP F36V -2xHA_P2A_BFP, DHX15-FKBP F36V -2xHA_P2A_HygR, and DHX38-FKBP F36V -2xHA_P2A_BFP were synthesized with 800bp dsDNA homology arms by Genewiz and assembled using the NEBuilder HiFi DNA assembly cloning kit (NEB E5520S).

Techniques: Construct

Journal: Cell reports

Article Title: Splicing quality control mediated by DHX15 and its G-patch activator SUGP1

doi: 10.1016/j.celrep.2023.113223

Figure Lengend Snippet: (A) Diagram of split-APEX (sAPEX) proximity labeling experiments with AP-tagged DHX15 and EX-tagged SUGP1. BP, biotin-phenol; APEX, ascorbate peroxidase. Upon short incubation of H2O2, DHX15-SUGP1 interaction-dependent reconstitution of APEX activity oxidizes BP into biotin-phenoxyl radicals, which then biotinylated proteins within a radius of several nanometers. (B) Protein blots (left) and quantification (right) of biotinylated proteins labeled by DHX15-SUGP1 interaction-reconstituted sAPEX activity. Ponceau S protein stain, loading control for total protein; streptavidin IRDye, detection of biotinylated proteins; FRB-nls, FRB control protein fused with an SV40 NLS. (C) Diagram of DHX15’s primary domain structure and sites of mutations tested in (E), (F), and (H). (D) Predicted protein complex structure of DHX15 interaction with SUGP1 G-patch domain by ColabFold.43 Arrows, sites of three mutations, and the corresponding functional centers. Colors of domains and mutations match (C). (E) Protein blots (left) and quantification (right) of biotinylated proteins labeled by WT versus mutant DHX15-SUGP1 interaction-reconstituted split-APEX activity. (F) Protein blots of biotinylated proteins labeled in (E), enriched by streptavidin-coated bead pull-down experiments. Cell lysates were collected after 24 h of AP-DHX15 and EX-SUGP1 co-transfection. WT and mutant AP-DHX15 expression induced by doxycycline addition for 24 h. (G) Quantification of pull-downs in (F). (H) Similar to (F), except that WT and mutant AP-DHX15 expressions were induced for 4 h. FLAG-AP-DHX15 detectable in the inputs on both anti-FLAG and anti-DHX15 blots. Red asterisk, FLAG-AP-DHX15 band; orange asterisk, endogenous DHX15 band. (I) Quantification of pull-downs in (H). (J) Model. Top row, hDHX15/ctPrp43 at ATP-bound open (PDB: 5ltk), G-patch domain-bound semi-open (PDB: 6sh6), and ADP-bound closed (PDB: 5dou) states. Middle row, side view of the open, semi-open, and closed states. Bottom row, cartoon representations of the structures, the G-patch domain (pink) binds to DHX15 at a semi-open state.

Article Snippet: DNA repair template plasmids containing DHX15-FKBP F36V -2xHA_P2A_BFP, DHX15-FKBP F36V -2xHA_P2A_HygR, and DHX38-FKBP F36V -2xHA_P2A_BFP were synthesized with 800bp dsDNA homology arms by Genewiz and assembled using the NEBuilder HiFi DNA assembly cloning kit (NEB E5520S).

Techniques: Labeling, Incubation, Activity Assay, Staining, Functional Assay, Mutagenesis, Cotransfection, Expressing

Journal: Cell reports

Article Title: Splicing quality control mediated by DHX15 and its G-patch activator SUGP1

doi: 10.1016/j.celrep.2023.113223

Figure Lengend Snippet: (A) Diagram of SUGP1’s primary domain structure. (B) Protein blots and quantification of biotinylated proteins labeled by FL versus truncated SUGP1-DHX15 split-APEX activity. Ponceau S protein stain, loading control for total protein; streptavidin IRDye, detection of biotinylated proteins; FRB-nls, FRB control protein fused with an SV40 NLS; ΔGp, G-patch domain truncation; ΔULM, ULM truncation; ΔS1&2, SURP1 and SURP2 truncation; colored asterisks, corresponding bands to FL and truncated HA-SUGP1-EX. (C) Protein blots of biotinylated proteins labeled in (B) enriched by streptavidin pull-down experiments. (D) Quantification of pull-downs in (C). (E) Fluorescent microscopy images of biotinylation signals (streptavidin, magenta), antibodies detecting HA-tagged SUGP1-EX, FL versus truncations (HA, red), FLAG-tagged AP-DHX15 (FLAG, green), nuclear DNA dye (DAPI, blue), and merged channels. (F) Fluorescent microscopy images of antibody detecting HA-tagged SUGP1.W387A-EX (HA, red), nuclear DNA dye (DAPI, blue), and merged channels. (G) Protein blots and quantification of biotinylated proteins labeled by full-length (FL), ΔULM, versus W387A mutant SUGP1-DHX15 interaction-reconstituted split-APEX activity. Ponceau S protein stain, loading control for total protein; streptavidin IRDye, detection of biotinylated proteins. Biotinylated HA was detected by merging the streptavidin channel (green) with the HA channel (red). See also Figure S4.

Article Snippet: DNA repair template plasmids containing DHX15-FKBP F36V -2xHA_P2A_BFP, DHX15-FKBP F36V -2xHA_P2A_HygR, and DHX38-FKBP F36V -2xHA_P2A_BFP were synthesized with 800bp dsDNA homology arms by Genewiz and assembled using the NEBuilder HiFi DNA assembly cloning kit (NEB E5520S).

Techniques: Labeling, Activity Assay, Staining, Microscopy, Mutagenesis

Journal: Cell reports

Article Title: Splicing quality control mediated by DHX15 and its G-patch activator SUGP1

doi: 10.1016/j.celrep.2023.113223

Figure Lengend Snippet: (A) Protein blots and quantification of biotinylated proteins labeled by FL versus truncated SUGP1-DHX15 interaction-reconstituted split-APEX activity. Ponceau S protein stain, loading control for total protein; streptavidin IRDye, detection of biotinylated proteins; FRB-nls, FRB control protein fused with an SV40 NLS; ΔGp, G-patch domain truncation; Gp-nls, G-patch domain alone fused with an SV40 NLS. (B) Fluorescent microscopy images of biotinylation signals (streptavidin, magenta), antibodies detecting HA-tagged SUGP1-EX, FL versus Gp-nls (HA, red), FLAG-tagged AP-DHX15 (FLAG, green), nuclear DNA dye (DAPI, blue), and merged channels. (C) A sampling-and-recruitment model of DHX15-SUGP1 interaction during early-splicing QC.

Article Snippet: DNA repair template plasmids containing DHX15-FKBP F36V -2xHA_P2A_BFP, DHX15-FKBP F36V -2xHA_P2A_HygR, and DHX38-FKBP F36V -2xHA_P2A_BFP were synthesized with 800bp dsDNA homology arms by Genewiz and assembled using the NEBuilder HiFi DNA assembly cloning kit (NEB E5520S).

Techniques: Labeling, Activity Assay, Staining, Microscopy, Sampling

Journal: Cell reports

Article Title: Splicing quality control mediated by DHX15 and its G-patch activator SUGP1

doi: 10.1016/j.celrep.2023.113223

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: DNA repair template plasmids containing DHX15-FKBP F36V -2xHA_P2A_BFP, DHX15-FKBP F36V -2xHA_P2A_HygR, and DHX38-FKBP F36V -2xHA_P2A_BFP were synthesized with 800bp dsDNA homology arms by Genewiz and assembled using the NEBuilder HiFi DNA assembly cloning kit (NEB E5520S).

Techniques: Recombinant, Protease Inhibitor, Magnetic Beads, Clone Assay, Isolation, CRISPR, Software, Imaging

Journal: Molecular Cell

Article Title: ZNF143 is a transcriptional regulator of nuclear-encoded mitochondrial genes that acts independently of looping and CTCF

doi: 10.1016/j.molcel.2024.11.031

Figure Lengend Snippet: Generation and validation of a system for rapid depletion of ZFP143 in mESCs (A) Schematic representation of the dTAG degron system. The endogenous Zfp143 locus is modified to create a fusion with the FKBP12 F36V -2xHA-P2A-eGFP cassette. (B) Western blot analysis of ZFP143 levels with an antibody against the endogenous ZFP143 at different time points after dTAG-V1 treatment. (C) Western blot analysis of ZFP143 levels with an antibody against the HA tag at different time points after dTAG-V1 treatment. (D) Tornado plots showing ZFP143-HA ChIP-seq signal centered at ZFP143-HA peaks in DMSO- and dTAG-V1-treated cells. (E) Genomic tracks showing ZFP143-HA ChIP-seq (red) and TT-seq nascent transcription (yellow for sense and purple for antisense transcription) at Rpp30 and Pomgnt2 in DMSO-treated and dTAG-V1-treated cells. (F) Fraction of ZFP143-bound genes among downregulated (green), upregulated (pink), and stable (gray) genes in the TT-seq at different time points after ZFP143 depletion.

Article Snippet: The DNA sequence for the knockin was designed to include the FKBP-2xHA-P2A-GFP cassette in between two homology arms for surrounding the STOP codon of Znf143 gene and ordered from Twist Bioscience.

Techniques: Modification, Western Blot, ChIP-sequencing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Tcl1 protein functions as an inhibitor of de novo DNA methylation in B-cell chronic lymphocytic leukemia (CLL)

doi: 10.1073/pnas.1200003109

Figure Lengend Snippet: Tcl1 interacts with Dnmt3A and Dnmt3B. (A) (Left) HEK 293 cells were cotransfected with DNMT3A-FLAG and 2xHA-TCL1 or with DNMT3A-FLAG and 2xHA-FHIT as indicated. After lysis, immunoprecipitation was carried out using anti-FLAG, IgG, or anti-HA antibodies. Western blot analysis was performed as indicated. (Right) HEK 293 cells were cotransfected DNMT3A-FLAG and Omni-GST-TCL1 or with DNMT3A-FLAG and Omni-GST-FHIT as indicated. After lysis and GST pulldown, Western blot analysis was performed as indicated. (B) Same as A, except using DNMT3B-FLAG instead DNMT3A-FLAG. (C) Daudi cells were lysed, and immunoprecipitation was carried out using anti-Tcl1, IgG, anti-Dnmt3A, or anti-Dnmt3B antibodies. Western blot analysis was performed as indicated. (D) HEK 293 cells were cotransfected with DNMT3A-FLAG and 2xHA-TCL1, 2xHA-FHIT, or 2xHA-TCL1b as indicated. After lysis, immunoprecipitation was carried out using anti-FLAG or anti-HA antibodies, and Western blot analysis was performed as indicated. (E) HEK 293 cells were cotransfected with Omni-GST-DNMT3B and 2xHA-TCL1, 2xHA-FHIT, or 2xHA-TCL1b as indicated. After lysis and GST pulldown, Western blot analysis was performed using anti-Omni (Top) or anti-HA (Middle and Bottom) antibodies. (F) Same as A, Left, but using DNMT1-FLAG instead of DNMT3A-FLAG. (G) Same as A, Left, but using DNMT3L-FLAG instead DNMT3A-FLAG.

Article Snippet: Full-length human TCL1 , TCL1B , and FHIT ORFs were cloned into pCMV-2xHA vector [based on pCMV-HA vector (BD Biosciences), with an added HA tag, creating HA tags at both 5′ and 3′ termini of all ORFs].

Techniques: Lysis, Immunoprecipitation, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Tcl1 protein functions as an inhibitor of de novo DNA methylation in B-cell chronic lymphocytic leukemia (CLL)

doi: 10.1073/pnas.1200003109

Figure Lengend Snippet: Tcl1 inhibits Dnmt3a enzymatic activity. (Upper) HEK 293 cells were cotransfected with DNMT3A-FLAG and either 2xHA-TCL1 or vector construct. HEK 293 cells transfected only with vector were used as a negative control. Cell lysates were immunoprecipitated for 2 h with anti-FLAG. Dnmt3a enzymatic activity was measured as described in Materials and Methods. (Lower) Amounts of Dnmt3A and Tcl1 in Dnmt3a activity assay were measured by Western blot analysis using anti-HA and anti-FLAG antibodies as indicated. The experiment was carried out in duplicate. Lanes 1 and 2 correspond to the left bar, lanes 3 and 4 correspond to the middle bar, and lanes 5 and 6 correspond to the right bar.

Article Snippet: Full-length human TCL1 , TCL1B , and FHIT ORFs were cloned into pCMV-2xHA vector [based on pCMV-HA vector (BD Biosciences), with an added HA tag, creating HA tags at both 5′ and 3′ termini of all ORFs].

Techniques: Activity Assay, Plasmid Preparation, Construct, Transfection, Negative Control, Immunoprecipitation, Western Blot