EMBO/soda-nel huggingface dataset:
TECRL, a new life-threatening inherited arrhythmia gene associated with overlapping clinical features of both LQTS and CPVT (2016)
Figure 3

Figure 3. Generation of hiPSCs and differentiation to cardiomyocytes.

(A) Skin fibroblasts (left) from IV:13, homozygous for the TECRLc.331+1G>A mutation were reprogrammed to hiPSCs (center), which expressed the pluripotency markers NANOG and SSEA4 (right). Scale bars: 100 μm. (B) TECRLHom-hiPSCs have a normal karyotype (left) and PluriTest demonstrates a high pluripotency score and low novelty score for all three hiPSC lines (right). (C) TECRLHom-hiPSCs generate derivatives of mesoderm (left), endoderm (center) and ectoderm (right). Scale bars: 25 μm (D) DNA sequencing confirms TECRLc.331+1G>A in the affected family members. Control (left); Heterozygous (Center); Homozygous (right). (E) Schematic of the protocol used for cardiac differentiation of hiPSCs. (F) ACTN2 immunostaining of CTRL-, TECRLHet- and TECRLHom-hiPSC-CMs. Scale bar: 25 μm. (G) Analysis of RT-PCR products by gel electrophoresis. Amplification products of TECRL exons 2-4 from CTRL-, TECRLHet- and TECRLHom-hiPSC-CMs (left) and coding sequence of TECRL from CTRL- and TECRLHom-hiPSC-CMs (right).
TECRL, a new life-threatening inherited arrhythmia gene associated with overlapping clinical features of both LQTS and CPVT (2016)
Figure 5

Figure 5. [Ca2+]i extrusion mechanisms in hiPSC-CMs.

(A) Representative traces of [Ca2+]i transients in Indo-1 AM-loaded hiPSC-CMs paced at 1 Hz, in the presence of caffeine (caff) or caffeine and NiCl2. (B) Amplitude of [Ca2+]i in the presence of caffeine and NiCl2 and fractional SR release in hiPSC-CMs. (C) Rate constants of SERCA-, NCX- and slow mechanisms-based [Ca2+]i decay in hiPSC-CMs. (D) Relative contribution of SERCA, NCX and slow mechanisms to [Ca2+]i extrusion in hiPSC-CMs.

Data information: n = 2-3 cells each from three independent experiments; *P<0.05 statistical significance was assessed with one-way ANOVA on ranks followed by Dunn’s test in case failed normality or variance. Otherwise, one-way ANOVA followed by Student-Neuman-Keuls test was used.
TECRL, a new life-threatening inherited arrhythmia gene associated with overlapping clinical features of both LQTS and CPVT (2016)
Figure 6

Figure 6. AP characteristics of TECRL-hiPSC-CMs.

(A) AP illustrating the analysed parameters (B) Representative APs from control (CTRL), heterozygous (HET) and homozygous (HOM) CMs. (C) RMP, dV/dtmax , APAmax, APAplat, APD20, APD50 and APD90 of CTRL-, TECRLHet- and TECRLHom-hiPSC-CMs.

Data information: n = 3-4 cells each from three independent experiments; *P<0.05 statistical significance was assessed with one-way ANOVA on ranks followed by Dunn’s test in case failed normality or variance. Otherwise, one-way ANOVA followed by Student-Neuman-Keuls test was used; AP = action potential; APAmax = maximum AP amplitude, APAplat = AP plateau amplitude; APD20, APD50, and APD90 = AP duration at 20, 50, and 90% repolarization, respectively; dV/dtmax = maximum upstroke velocity; RMP = resting membrane potential.
TECRL, a new life-threatening inherited arrhythmia gene associated with overlapping clinical features of both LQTS and CPVT (2016)
Figure 8

Figure 8. Flecainide alleviates triggered activity in TECRLHom-hiPSC-CMs.

(A) Representative AP trace of a TECRLHom-hiPSC-CM in the presence of NA alone or NA and flecainide. (B) Effect of flecainide on AP parameters of hiPSC-CMs. (C-D) Addition of 5 μM of flecainide decreased the susceptibility to triggered activity in hiPSC-CMs. Note that the effect of flecainide on triggered activity of TECRLHet- and TECRLHom-hiPSC-CMs is more pronounced than its effect on their spontaneous activity.

Data information: n = 1-2 cells each from three independent experiments; *P<0.05, statistical significance within a group before and after application of flecainide was assessed with paired t-test; NA = noradrenaline, Flec = flecainide
Parkinson's disease motor symptoms rescue by CRISPRa-reprogramming astrocytes into GABAergic neurons (2022)
Figure 1

Figure 1: The Rosa26 knock-in dCas9 Activator Mouse (dCAM)

A, Knock-in of a conditional dCas9-VPR-P2A-SAM expression cassette into the Gt(ROSA)26Sor locus enables flexible multiplexed endogenous gene activation in vitro and in vivo. The cassette is composed of a ubiquitous CAG promoter, a stop cassette (Stop LoxP-puro-stop-LoxP) followed by the FRT flanked SAM activator, a P2A peptide and dCas9-VPR. dCAM x Gfap-Cre mice enable astrocyte-specific dCas9 and activator expression. For in vivo activation, six sgRNAs, driven by different Pol III promoters (H1, hU6, mU6 and 7SK) and the marker gene FLEx-GFP, or split-FLEx-GFP respectively, driven by a CBh promoter are delivered by AAV.

B, Multiplexed activation of Ascl1, Lmx1a, NeuroD1 and of Ascl1, Lmx1a, Nr4a2 in primary astrocytic cultures. n=3, representative experiment from 2-3 independent experiments, additional data in supplement. Activation levels are depicted as fold change between cells transfected with and without sgRNAs. All levels were normalized to β-Actin. Error bars represent mean ± SD between technical replicates.

C, Experimental Outline: 6-hydroxydopamine (6-OHDA-HCl) is stereotactic injected into the medium forebrain bundle to induce nigrostriatal dopaminergic neurodegeneration. Two weeks later, an AAV expressing gRNAs and a fluorescent reporter is injected into the dorsal striatum. Animals were analyzed after 5 and 13 weeks post injection (wpi) including behavior tests, electrophysiological measurements and immunohistochemistry. Abbreviations: Puro - puromycin resistance, SAM - synergistic activation mediator (MS2: MS2 bacteriophage coat protein, p65: p65 subunit of human NF-ĸB, HSF1: Heat shock factor 1), P2A - 2A self-cleaving peptide, dCas9 - deadCas9 (nuclease-deficient), VPR - VP64: 4x VP16 herpes simplex virus protein vmw65, p65, Rta: Regulator of transcriptional activation, CAG - CMV early enhancer/chicken β actin promoter, CBh - chicken ß-actin hybrid promoter. SgRNA expression is driven by the different Pol III promoters (H1, hU6, mU6 and 7SK).
Parkinson's disease motor symptoms rescue by CRISPRa-reprogramming astrocytes into GABAergic neurons (2022)
Figure 2

Figure 2: dCas9 Activator Mouse (dCAM) based reprogramming of astrocytes

A, Representative photomicrographs taken from the dorsal striatum 13 weeks after AAV-injection. In mice injected with GFP control virus, virtually all GFP positive cells depict an astrocytic morphology, many GFP positive cells in ALNe-218 and ALN-treated animals show a neuron-like morphology.

B, Immunohistochemical analysis showing GFP+/GFAP+ double positive cells 13 wpi. Arrows indicate double positive GFP+/GFAP+ cells, arrowheads indicate GFP+/GFAP- cells. Quantification of GFAP+/GFP+ cells shows a significant decrease upon ALNe-218 and ALN-treatment (GFP vs. ALNe-218 P=0.0064, GFP vs. ALN P=0.0002 and ALN vs. ALNe-218 P=0.0127). Multiple comparison ANOVA F(2,7)=32.06.

C, Double immunostaining for GFP and the neuronal marker NeuN. Arrowheads indicate double positive GFP+/NeuN+ cell 13 wpi. Quantification demonstrate a significant increase in NeuN+/GFP+ cells upon ALNe-218 and ALN-induction (GFP vs. ALNe-218 P<0.0001, GFP vs. ALN P<0.0001 and ALN vs. ALNe-218 P=0.0012). Multiple comparison ANOVA F(2,6)=170.3.

D, Double immunostaining for GFP and TH as a marker for dopaminergic neurons in the dorsal striatum of GFP-control, ALNe-218 and ALN-treated animals. Arrowheads indicate GFP+/TH- cells with neuronal morphology. No GFP+/TH+ cell could be detected in any of the experimental groups. Scale bars indicate 20 µm.

Data information: Scale bars indicate 20 µm. Tukey's multiple comparisons test * P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. n = 3-4 mice per condition. Error bars represent mean ± SD.
Parkinson's disease motor symptoms rescue by CRISPRa-reprogramming astrocytes into GABAergic neurons (2022)
Figure 3

Figure 3: The AAV-split-dCas9 Activator System (AAV-dCAS).

A, dCas9 is separated into a N- and a C-terminal part (AAV-N-dCas9aa1-573-N-intein and AAV-C-dCas9aa574-1368-VP64-C-intein), both portions are fused to the corresponding intein-moieties. Upon co-expression intein-mediated trans-splicing leads to reconstitution of Cas9 protein.

B, RT-qPCR analysis of Ascl1 induction comparing the activation capacity of full-length versus split-dCas9 in Neuro2A cells (data in fold change normalized to non-activated control; dCas9 6116 ± 847.3, split-dCas9 4415 ± 748.8, n=3 biological replicates, box-and-whiskers plots indicate median, 25th-75th percentile and min/max whiskers).

C, Immunocytochemistry analysis of reprogrammed primary astrocytes cells 16 days after lentiviral transduction revealed successful in vitro reprogramming into neurons using CRISPRa. Astrocytes are infected with two lentiviruses expressing dCas9-VPR in a intein-split version similar to the AAV-dCAS system but driven by a Tet-O promoter. Tet-O driven dsRed and Ascl1 cDNA expressing construct were used as negative and positive controls respectively. Arrows indicate single MAP2 positive background neurons, arrowheads indicate double positive induced neurons. Scale bar indicates 20 µm.

D, Schematic representation of the AAV-dCAS system: For induction of up to five endogenous genes plus a GFP reporter, a total of four different AAVs are utilized. dCas9 is delivered by two AAVs, a third AAV is needed for the delivery of the SAM activator. A fourth virus contains the reporter gene, while sgRNAs are distributed between the vectors. To ensure cell type specificity upon Cre expression, the N-dCas9 and the GFP are inverted and flanked by two different LoxP sites (LoxX and Lox511).

E, Multiplexed activation of Ascl1/Lmx1a/NeuroD1, Ascl1/Lmx1a/Nr4a2 and Ascl1/ Lmx1a/ Nr4a2/Pitx3/FoxA2 in primary astrocytic cells. n=2-3 biological replicates, one representative run is shown, additional data in supplement. Activation levels are depicted as fold change between cells transfected with and without sgRNAs. All levels were normalized to β-Actin. Error bars represent mean ± SD between technical replicates.

Data information: Abbreviations: dN-Cas9 - N-terminal dCas9-residues 1-573, N-intein - N-terminal part of DNA polymerase III subunit alpha, dC-Cas9 - C-terminal dCas9 residues 574-1368, C-intein - C-terminal part of DNA polymerase III subunit alpha, VP64 - 4x VP16 herpes simplex virus protein vmw65, p65 - p65 subunit of human NF-ĸB, HSF1 - heat shock factor 1, MS2 - MS2 bacteriophage coat protein, PAM - protospacer adjacent motif, TSS - transcriptional start site, OE - overexpression, CBh - chicken β-actin hybrid promoter. SgRNA expression is driven by the different Pol III promoters (H1, hU6, mU6 and 7SK).
Parkinson's disease motor symptoms rescue by CRISPRa-reprogramming astrocytes into GABAergic neurons (2022)
Figure 4

Figure 4: AAV-split-dCas9 Activator System (AAV-dCAS) based reprogramming of astrocytes.

A, Representative photomicrographs taken from the dorsal striatum 13 weeks after AAV-injection. In the GFP control condition, virtually all GFP positive cells depict an astrocytic morphology, many GFP positive cells in ALNe-218 and ALN-treated animals show a neuron-like morphology.

B, Immunohistochemical analysis showing GFP+/GFAP+ double positive cells 13 wpi. Arrows indicate double positive GFP+/GFAP+ cells, arrowheads indicate GFP+/GFAP- cells. Quantification of GFAP+/GFP+ cells shows a significant decrease upon ALNe-218 and ALN-treatment (GFP vs. ALNe-218 P=0.0083, GFP vs. ALN P<0.0001 and ALN vs. ALNe-218 P=0.0006, multiple comparison ANOVA F(2,6)=79.76).

C, Photomicrographs showing GFP+/NeuN+ neurons 13 wpi. Arrowheads indicating GFP+/NeuN+ induced neurons. Quantification demonstrate a significant increase in NeuN+/GFP+ cells upon ALNe-218 and ALN-induction (GFP vs. ALN P=0.0008, ALN vs. ALNe-218 P=0.0092, multiple comparison ANOVA F(2,7)=21.74).

D, In ALN-induced neurons, confocal analysis is demonstrating a co-localization of GFP and glutamic acid decarboxylase (Gad65/67), a marker specific for GABAergic neurons.

E, Quantification of GAD6567+/GFP+ cells with neuronal morphology shows that the majority of ALN-induced neurons are colocalizing with this GABAergic marker both in the AAV-dCAS as well as in the dCAM setting.

Data information: Scale bars indicate 20 µm. Error bars represent mean ± SD. n = 3-4 mice (b,c) and n= 6-8 mice (e) per condition. Tukey's multiple comparisons test * P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
Identification of an oncogenic network with prognostic and therapeutic value in prostate cancer (2018)
Figure 1

Figure 1: Multi-faceted system-level analyses identifies seven prostate cancer hub genes. (A) Module-trait relationships were established by WGCNA using eight independent microarray analyses comprising 375 human prostate samples. Gene modules (y axis) are denoted by an arbitrary color name. Bins show the Pearson correlation value between gene expression levels of each module within the noted phenotype/disease stage (x axis) and p values. A value of 1 (red) quantifies the strongest positive correlation (genes are upregulated), -1 (blue) the strongest negative correlation (genes are downregulated), and 0 (white) no correlation.

(B) Microarray analysis was performed following doxycycline-regulated specific AR-V7 depletion (using a tet-plKO backbone) in 22Rv1 PC cells compared to doxycycline-treated shGFP controls. The genes that were significantly regulated by shAR-V7 (in either direction, p value < 0.05) were distributed among the gene modules defined by WGCNA in panel A. Upregulated genes (red) are those in which expression decreased following AR-V7 depletion and conversely downregulated genes (blue) are those that increased following AR-V7 depletion.

(C) AR-V7 human functional interactome was generated using SGA screening in the yeast S. pombe, combined with STRING data to map protein-protein interactions, followed by the identification of the human orthologs. The colors denote the different types of genetic interactions: red are genes that when deleted in yeast and crossed with AR-V7-expressing yeast were synthetic lethal while green denotes genes that caused a growth suppression. White designates yeast essential genes (ie: genes that are critical for yeast survival, and thus could not be present in the yeast deletion library), but were incorporated into the network based on the criteria that they are known (based on literature) to physically interact with at least two of the red or green genes. Pink are a combination of essential and nonessential genes identifying the same human protein.

(D) Table summarizing the seven PC hub genes identified by the system-level analyses.

(E) Network interactions of the seven genes with the 50 most frequently altered neighbor genes, were mapped using cbioportal.org. The type of gene to gene interactions are: controls state change of (blue), controls expression of (green), in complex with (brown).
Identification of an oncogenic network with prognostic and therapeutic value in prostate cancer (2018)
Figure 3

Figure 3: Depletion of the expression of members of the seven gene set reduces CRPC cell proliferation and AR ligand-independent transcriptional activity.

(A) Cell proliferation was examined in the CRPC cell line 22Rv1 following individual depletion of mRNAs for the seven genes or shGFP controls, using shRNA against the coding region for each gene (shRNA #2). Cell number was measured using a non-perturbing nuclear restricted dye and quantified after 72 hours using Incucyte Zoom System. Data shown are mean ± s.e.m. of 8 to 12 replicates normalized to their shGFP control. Kruskal-Wallis test (p value < 0.0001, two-tailed) and Dunn's multiple comparisons test were performed.

(B) Representative images of 22Rv1 stably depleted of BUB1B, or control (shGFP) are shown.

(C) 22Rv1 stably depleted of each of the seven genes were transfected with a dual plasmid luciferase reporter system which quantifies AR activity and basal transcription. The assay was conducted in 2% CSS to measure AR ligand-independent transcriptional activity. Data represent two independent experiments performed in triplicate, showing the mean ± s.e.m., and normalized to their shGFP controls. Kruskal-Wallis test (p value < 0.0001, two-tailed) and Dunn's multiple comparisons test were performed. * Significant at a p value < 0.05, ** p value < 0.001.

(D), (E) The expression of FKBP5 and UBE2C determined by RT-qPCR analysis and normalized to GAPDH mRNA levels was examined in 22Rv1 cells stably expressing shRNA for each of the seven genes. Cells were cultured in 2% CSS. Data represent two independent experiments performed in duplicate or triplicate, showing the mean ± s.e.m., and normalized to their shGFP controls. Kruskal-Wallis test (p value < 0.0001, two-tailed) and Dunn's multiple comparisons test were performed. * Significant at a p value < 0.05, ** p value < 0.001.
m6A-mediated alternative splicing coupled with nonsense-mediated mRNA decay regulates SAM synthetase homeostasis (2021)
Figure 1

Figure 1. Long-read sequencing reveals putative NMD isoforms enriched in the smg-2 mutant.

(A) Venn diagram of exon-exon junctions in 6,642 common genes identified among the Nanopore reads from the smg-2 (yb979) mutant and N2 as well as those deduced from gene models in WormBase (WS271).

(B) Volcano plot of 8,701 mRNA isoforms from 2,931 genes with multiple isoforms. X-axis indicates a difference in percentage of each mRNA isoform within the gene (Δ%smg-2-N2). Y-axis indicates -log10 of p-value in Fisher's exact tests. Blue symbols indicate 219 mRNA isoforms significantly depleted from the smg-2 mutant (p<0.05, Δ%smg-2-N2<-10). Red symbols indicate 420 isoforms significantly enriched in the smg-2 mutant (p<0.05, Δ%smg-2-N2>10).

(C, D) Length distribution of 3'UTRs (C) and proportions of putative PTC-containing isoforms (D) in 10,136 mRNA isoforms detected in N2 and/or smg-2 (All), the 219 isoforms depleted from and the 420 isoforms enriched in the smg-2 mutant.

(E) GO terms significantly enriched in 259 genes with smg-2-enriched PTC isoforms (top) and in 30 genes with smg-2-depleted PTC isoforms (bottom). Modified Fisher's exact p-values in the DAVID Bioinformatics system are indicated. Note that 14 of the 30 genes with the smg-2-depleted mRNA PTC isoforms are common with those having smg-2-enriched PTC isoforms. This is why the same GO terms are enriched in these two groups. The mRNAs from such genes likely contain upstream open reading frames (uORFs), which may not be directly involved in or differentially affect NMD.
m6A-mediated alternative splicing coupled with nonsense-mediated mRNA decay regulates SAM synthetase homeostasis (2021)
Figure 2

Figure 2. Alternative splicing of sams-3, sams-4 and sams-5 are dynamically regulated upon feeding.

(A) Schematic representation of the sams genes in C. elegans. Boxes indicate exons. Coding regions of productive mRNAs are in orange. A truncated coding region with a premature termination codon in a pseudogene sams-2 is colored cyan. Double arrows illustrate regions deleted in mutant alleles indicated.

(B) Alternative splicing of sams-3, sams-4 and sams-5 in unfed and fed worms without or with a protein synthesis inhibitor emetine. Synchronized L1 larvae of an NMD-deficient strain KH1668: smg-2 (yb979) (lanes 1-3) and a wild-type strain N2 (lanes 4-6) were incubated in S-complete medium alone (lanes 1, 4), with a standard E. coli strain OP50 (OD600=10.0) (lanes 2, 5) or OP50 supplemented with 10 mg/ml emetine (lanes 3, 6) for 3 hours at 20°C. Total RNAs were extracted from whole animals and subjected to semi-quantitative RT-PCR, whose products were analyzed by capillary electrophoresis. Representative gel-like presentation is indicated (n=3). Schematic structure of each PCR product is indicated on the right. Open reading frames (ORFs) for full-length and truncated proteins are in orange and cyan, respectively. rpl-12 was used as an unaffected control.
m6A-mediated alternative splicing coupled with nonsense-mediated mRNA decay regulates SAM synthetase homeostasis (2021)
Figure 3

Figure 3. Expression of sams-3, sams-4 and sams-5 is negatively regulated by SAM synthetase activity through alternative splicing.

(A) Alternative splicing of sams-3, sams-4 and sams-5 in smg-2 (yb979) and smg-2 (yb979); sams-5 (gk147); sams-1 (ok2946) mutants. Synchronized L1 larvae of each strain were incubated in S-complete medium alone (lanes 1 and 5), with OP50 (OD600=10.0) (lanes 2 and 6) and with OP50 supplemented with 25 mM L-Met (lanes 3 and 7) or 25 mM cycloleucine (cLeu) (lanes 4 and 8) for 3 hours at 20°C. The splicing patterns were analyzed and presented as in Figure 2B (n=3).

(B, C) Western blot analysis of SAMS-1, SAMS-3 and SAMS-4 during larval development in the smg-2 (yb979) (B) and wild-type (C) backgrounds. Genotypes of the worms are smg-2 (w) and smg-2; sams-5; sams-1 (s) in (B) and wild-type (w) and sams-1 (s) in (C). Synchronized L1 larvae of each strain were incubated with OP50 at 20°C and subjected to Western blot analysis at indicated time points. Anti-β-tubulin (B) or coomassie brilliant blue (CBB) staining (C) was used as a loading control. Specificity of the antibodies is confirmed in Appendix Figure S12. Note that upregulation of SAMS-1 protein in the wild type during larval development in (C) is consistent with feeding-induced upregulation of sams-1 mRNA in Figure EV3D.
m6A-mediated alternative splicing coupled with nonsense-mediated mRNA decay regulates SAM synthetase homeostasis (2021)
Figure 4

Figure 4. mett-10 is required for effective production of NMD isoforms from the sams genes in vivo.

(A) Nucleotide sequence alignment of putative stem-loop structures in the C. elegans sams-3, sams-4 and sams-5 genes together with six hairpin structures in 3'UTR of the human MAT2A gene. Conserved residues are shaded in black. Loop, transition and stem regions of the MAT2A hairpins (Doxtader et al, 2018) are boxed in magenta, green and grey, respectively. An arrowhead indicates adenine bases in the MAT2A hairpins that are specifically modified into m6A by METTL16 in vitro (Shima et al, 2017).

(B) Schematic representation of the predicted hairpin structures in sams-3/sams-4 (left) and sams-5 (right) pre-mRNAs. The boundaries between intron 2 and exon 3 are indicated. Arrowheads indicate the m6A modification sites.

(C) Alternative splicing of sams-3, sams-4 and sams-5 in smg-2 (yb979) and smg-2 (yb979); mett-10 (ok2204) mutants. Synchronized L1 larvae of each strain were incubated in S-complete medium alone (lanes 1 and 3) or with OP50 (lanes 2 and 4) for 3 hours at 20°C and the splicing patterns were analyzed and presented as in Figure 2B.
m6A-mediated alternative splicing coupled with nonsense-mediated mRNA decay regulates SAM synthetase homeostasis (2021)
Figure 5

Figure 5. METT-10 catalyzes m6A modification at the invariant AG dinucleotide of the distal 3'SS in vitro.

(A) Mass chromatograms of RNA fragments to detect m6A (top) or unmodified A (bottom) after in vitro incubation of 127-nt sams-3/sams-4 pre-mRNA (sequence available in Fig EV2) with recombinant full-length METT-10 protein in the presence (left) or absence (right) of 1 mM SAM. The sequence, m/z value, and charge state for each fragment are indicated on the right. Asterisks indicate non-specific signals.

(B) The negatively-charged ions of RNase T1 fragment is decomposed in the instrument by collision-induced dissociation (CID) using helium gas. The product ions produced by CID are assigned on the sequence illustrated on the top right inset panel. Nomenclature of the product ions are described in the literature (McLuckey et al, 1992). Product ions of c and y series derive from the 5' and 3' termini of the fragment, respectively.