large rnas  (Zymo Research)

Journal: bioRxiv

Article Title: RNA G-Quadruplexes Function as a Tunable Switch of FUS Phase Separation

doi: 10.1101/2025.10.31.685846

Figure Lengend Snippet: (A) Schematic of experimental paradigms. FUS phase separation is initiated by adding TEV protease to cleave MBP tag from MBP-TEV-FUS. For inhibition experiments, RNA is added to FUS phase separation reactions prior to initiating FUS phase separation with TEV protease. For reversal experiments, FUS phase separation is initiated by TEV protease 60 minutes prior to adding RNA. (B) Representative images of FUS droplets formed without RNA. LLPS of FUS (1.8 µM FUS supplemented with 0.2 µM FUS-GFP) was monitored by droplet imaging at indicated times. Scale bar is 5 µm. The images without RNA presented in were collected in the same trial as the data presented in . ( C, H ) Phase diagrams summarizing the inhibition of FUS LLPS by RNA S1 ( C ) or W1 ( H ). Average droplet count defines symbol size, and average FUS enrichment score (quantified by dividing mean GFP signal within droplets by mean GFP signal in background) defines symbol saturation. Data used to generate phase diagrams are the mean of three independent experiments. ( D, G ) Representative images of FUS droplets formed in the presence of RNA S1 ( D ) or W1 ( G ). LLPS of FUS (1.8 µM FUS supplemented with 0.2 µM FUS-GFP) with RNA (1, 8, or 32 µM unlabeled RNA supplemented with 75 nM Cy5-RNA) was monitored by droplet imaging every 30 minutes for a duration of 150 minutes. 120-minute timepoint images were shown due to space limitations to represent RNA effect on FUS LLPS. Scale bar is 5 µm. ( E, I ) Turbidity measurement of FUS LLPS inhibition by RNA S1 ( E ) or W1 ( I ). MBP-FUS (2 µM) was incubated in the presence or absence of RNA prior to initiating LLPS reaction with TEV protease. Turbidity at 395 nm was monitored at 25°C over 180 min. Data represent mean±SEM (n=3-4). The 0 µM RNA control plotted in , , and S1C are the same because these experiments were conducted simultaneously. ( F, J ) Area under the curve (AUC) was calculated from turbidity curves in ( F ) and ( J ). Data represent mean±SEM (n=3-4). ( J ) Ordinary one-way ANOVA with Tukey’s multiple comparisons test was used to compare the means between RNA W1 concentrations (ns P>0.05, **P≤0.005). ( K, O ) Representative images of FUS droplets 60 minutes after the addition of RNA S1 ( K ) or W1 ( O ). FUS (1.8 µM FUS supplemented with 0.2 µM FUS-GFP) droplets were formed for 60 minutes prior to adding RNA (1, 8, or 32 µM unlabeled RNA supplemented with 75 nM Cy5-RNA). Droplets were monitored by imaging immediately before RNA, then every 30 minutes for a duration of 120 minutes after RNA was added. The 60-minute post-RNA timepoint images were selected to represent RNA’s effect on FUS LLPS. Scale bar is 5 µm. ( L, P ) Phase diagrams summarizing the reversal of FUS LLPS by RNA S1 ( L ) or W1 ( P ). Droplet images collected in ( L ) or 1O ( P ) were quantified. Average droplet count defines symbol size and average FUS enrichment score defines symbol saturation. Data used to generate phase diagrams are the mean of three independent experiments. ( M, Q ) Turbidity measurement of FUS LLPS reversal with RNA S1 ( M ) or W1 ( Q ). FUS (2 µM) LLPS was initiated, and monitored by turbidity at 395 nm. After 60 min, data collection was paused to add RNA then resumed to monitor turbidity changes for an additional 120 min. Data represent mean±SEM (n=3). The 0 µM RNA control plotted in and are the same because these experiments were conducted simultaneously. ( N, R ) AUC was calculated from turbidity curves in ( N ) and ( R ) after RNA was added. Data represent mean±SEM (n=3). ( R ) Ordinary one-way ANOVA with Tukey’s multiple comparisons test was used to compare the means between RNA W1 concentrations (ns P>0.05, ****P<0.0001). ( S ) Microinjection of RNA S1 (100 µM) into HEK 293T cells expressing FUS-WT-eGFP. Panel 1 shows pre-injection condensates 1 (C1), 2 (C2, red arrows), and 3 (C3, yellow arrows). Panels 2-3 show suction pressure, where C2 flows into the pipette tip. Panels 4-6 show ejection pressure (∼10 kPa), where C2 is blocking RNA injection. Panels 7-8 show C2 and C3 dissolving after RNA S1 injection. Panel 9 is after stopping RNA injection.

Article Snippet: The Large RNA fraction (S1*10) was concentrated using RNA Clean & Concentrator-5 Kit (Zymo, R1013).

Techniques: Inhibition, Imaging, Incubation, Control, Microinjection, Expressing, Injection, Transferring, Blocking Assay

Journal: bioRxiv

Article Title: RNA G-Quadruplexes Function as a Tunable Switch of FUS Phase Separation

doi: 10.1101/2025.10.31.685846

Figure Lengend Snippet: (B) Representative images of FUS droplets before (“pre-RNA”, top) and after the addition of RNA S1 and/or S1_G4neg in TAB-K + . LLPS of FUS (1.8 µM FUS supplemented with 0.2 µM FUS-GFP) was monitored by droplet imaging before and 5 minutes after the addition of RNA. For “Single RNA”, S1 (4 µM + 75 nM Cy3-S1) or S1_G4neg (4 µM + 75 nM Cy5-S1_G4neg) were individually added to pre-formed FUS droplets. For “Mixed RNA”, S1 (2 µM + 75 nM Cy3-S1) and S1_G4neg (2 µM + 75 nM Cy5-S1_G4neg) were added to pre-formed FUS droplets. Scale bar is 5 µm. (C) Average RNA enrichment score within FUS droplets was quantified for the post-RNA images from . Bars represent mean±SEM and data points represent individual image averages (n=20) across two independent trials. Ordinary one-way ANOVA with Šidák’s multiple comparisons test was used to compare the means of different RNA (ns P>0.05, ****P<0.0001). (D) Intersection of FUS phase separation-dependent interactome (Droplet or Soluble) and the transcriptome-wide rG4 database . Overlaps with high probability of forming rG4 are in dark blue, those with medium probability are in blue, and those with low probability are in light blue. (E) Venn diagram of the high-probability rG4 sequences that overlap between FUS Droplet interactome, FUS Soluble interactome, and/or FUS CLIP-seq interactome. Indicated RNA sequences were selected from this analysis and used in and Fig. S7F-I. (F) Turbidity measurement of FUS LLPS reversal with RNA in TAB-K + . LLPS of FUS (2 µM) was initiated, and monitored by turbidity at 395 nm. After 90 min, data collection was paused to add RNA (32 µM). Data represent mean±SEM (n=3-7). (G) AUC was calculated from turbidity curves in after RNA was added. Data represent mean±SEM (n=3-7). Ordinary one-way ANOVA with Tukey’s multiple comparisons test was used to compare the means between RNAs (ns P>0.05, *P≤0.05, **P≤0.005, ****P<0.0001). (H) Representative images of FUS droplets before (“pre-RNA”) and after the addition of indicated RNAs. LLPS of FUS (1.8 µM FUS supplemented with 0.2 µM FUS-GFP) was monitored by droplet imaging before and 60 minutes after the addition of RNA (32 µM unlabeled RNA). Scale bar is 5 µm. (I) Turbidity measurement of FUS disaggregation with indicated rG4 RNAs and non-rG4 RNA controls. FUS (5 µM) aggregation was initiated by adding TEV protease to GST-TEV-FUS in Assembly Buffer and monitored by turbidity at 395 nm. After 120 min, data collection was paused to add RNA (100 ng/µL). Data represent mean±SEM (n=3). (J) AUC was calculated from turbidity curves in after RNA was added. Data represent mean±SEM (n=3). Ordinary one-way ANOVA with Tukey’s multiple comparisons test was used to compare the means between the RNA treatments. (ns P>0.05, ****P<0.0001). (K) Representative differential interference contrast (DIC) images of FUS aggregates before and after the addition of RNA. Pre-formed FUS aggregates were assembled as in (H), and images were acquired immediately before (“pre-RNA”) or 100 min after addition of buffer or RNA (100 ng/µL). Scale bar is 10 µm.

Article Snippet: The Large RNA fraction (S1*10) was concentrated using RNA Clean & Concentrator-5 Kit (Zymo, R1013).

Techniques: Imaging

Journal: bioRxiv

Article Title: RNA G-Quadruplexes Function as a Tunable Switch of FUS Phase Separation

doi: 10.1101/2025.10.31.685846

Figure Lengend Snippet: (A) Representative CD spectra of RNA S1*2 collected at 25°C in indicated buffer. (B) Turbidity measurement of FUS LLPS reversal with S1*2 in TAB-Na + (blue traces) or TAB-K + (red traces). LLPS of FUS (2 µM) was initiated, and monitored by turbidity at 395 nm. After 90 min, data collection was paused to add RNA. Increasing concentration of RNA is indicated by a more saturated color. Data represent mean±SEM (n=3). (C) AUC was calculated from turbidity curves in after RNA was added. Data represent mean±SEM (n=3). Ordinary one-way ANOVA with Šidák’s multiple comparisons test was used to compare the means between TAB-Na + and TAB-K + at each RNA concentration (ns P>0.05, *P≤0.05, ***P≤0.0005). ( D, E ) CD spectra of RNA S1 and S1*2 were normalized by RNA length and re-plotted to compare spectra in K + ( D ) and Na + ( E ). ( F, H ) Turbidity measurement of FUS LLPS reversal with RNA S1 or S1*2 in TAB-K + ( F ) or TAB-Na + ( H ). LLPS of FUS (2 µM) was initiated, and monitored by turbidity at 395 nm. After 90 min, data collection was paused to add RNA. Increasing concentration of RNA is indicated by a more saturated color. S1 data are represented by filled symbols and S1*2 data are represented by open symbols. Data represent mean±SEM (n=3). ( G, I ) AUC was calculated from turbidity curves in ( G ) and ( I ) after RNA was added. Data represent mean±SEM (n=3). Ordinary one-way ANOVA with Šidák’s multiple comparisons test was used to compare the means between RNAs at comparable concentrations, accounting for the difference in length between S1 and S1*2 (ns P>0.05, *P≤0.05, **P≤0.005). (J) Representative images of FUS assemblies formed in the presence of RNA S1 (2 µM), S1*2 (1 µM), or S1*10 (200 nM). LLPS of FUS-GFP (500 nM) in TAB-K + was initiated by adding TEV protease to cleave the MBP tag of MBP-TEV-FUS-GFP in the presence of indicated RNAs. For each trial, images were acquired 120 minutes after TEV addition (n=3). Scale bar is 5 µm and arrows point to FUS assemblies. (K) The average number of FUS assemblies per image was quantified from images collected in . Bars represent mean±SEM of three independent experiments, large symbols represent trial means (n=3) and small datapoints represent individual image values (n=30). Ordinary one-way ANOVA with Tukey’s multiple comparisons test was used to compare the means between RNAs. (ns P>0.05, *P≤0.05, ****P<0.0001).

Article Snippet: The Large RNA fraction (S1*10) was concentrated using RNA Clean & Concentrator-5 Kit (Zymo, R1013).

Techniques: Circular Dichroism, Concentration Assay

Journal: bioRxiv

Article Title: RNA G-Quadruplexes Function as a Tunable Switch of FUS Phase Separation

doi: 10.1101/2025.10.31.685846

Figure Lengend Snippet: ( A, G ) Representative circular dichroism (CD) spectra of RNA S1 ( A ) or S1_G4neg ( G ) collected at 25°C in indicated buffer condition. ( B ) Schematic representation of predicted rG4 formed by RNA S1. RNA structures were created with Biorender.com . ( C ) CD thermal melt curves of RNA S1 in Na + (blue) or K + (red). CD signals were collected at 265 nm from 15°C to 85°C. Data plotted represent mean±SEM (n=3). Black dashed lines represent the fitted curve used to obtain T m . ( D, H ) Turbidity measurement of FUS LLPS reversal with RNA S1 ( D ) or S1_G4neg ( H ) in TAB-Na + (blue traces) or TAB-K + (red traces). LLPS of FUS (2 µM) was initiated, and monitored by turbidity at 395 nm. After 90 min, data collection was paused to add RNA. Increasing concentration of RNA is indicated by a more saturated color. Data represent mean±SEM (n=3). The 0 µM RNA control in TAB-K + plotted in and Fig. S2F are the same because these experiments were conducted simultaneously. ( E, I ) AUC was calculated from turbidity curves in ( E ) or ( I ) after RNA was added. Data represent mean±SEM (n=3). Ordinary one-way ANOVA with Šidák’s multiple comparisons test was used to compare the means between TAB-Na + and TAB-K + at each RNA concentration (ns P>0.05, **P≤0.005, ***P≤0.0005, ****P<0.0001). ( F, J ) Representative images of FUS droplets before (top row) and after (bottom row) the addition of RNA S1 ( F ), or S1_G4neg ( J ). LLPS of FUS (1.8 µM FUS supplemented with 0.2 µM FUS-GFP) was monitored by droplet imaging before and 30 minutes after the addition of RNA (4 or 8 µM unlabeled RNA supplemented with 75 nM Cy5-RNA). Scale bar is 5 µm.

Article Snippet: The Large RNA fraction (S1*10) was concentrated using RNA Clean & Concentrator-5 Kit (Zymo, R1013).

Techniques: Circular Dichroism, Concentration Assay, Control, Imaging

Journal: bioRxiv

Article Title: RNA G-Quadruplexes Function as a Tunable Switch of FUS Phase Separation

doi: 10.1101/2025.10.31.685846

Figure Lengend Snippet: ( A, B ) CD thermal melt curves of RNA S1 and S1 P+M in K + ( A ) or Na + ( B ). Data plotted represent mean±SEM (n=3). Black dashed lines represent the fitted curve used to obtain T m . Thermal melt curves of RNA S1 are replotted from in order to compare with RNA S1 P+M . Extra sum-of-squares F Test was used to compare T m of S1 and S1 P+M in each buffer condition. (****P<0.0001). ( C, E ) Turbidity measurement of FUS LLPS reversal with RNA S1 or S1 P+M in TAB-K + ( C ) or TAB-Na + ( E ). LLPS of FUS (2 µM) was initiated, and monitored by turbidity at 395 nm. After 90 min, data collection was paused to add RNA. Increasing concentration of RNA is indicated by a more saturated color. S1 data are represented by filled symbols, and S1 P+M data are represented by open symbols. Data represent mean±SEM (n=3). ( D, F ) AUC was calculated from turbidity curves in ( D ) and ( F ) after RNA was added. Data represent mean±SEM (n=3). Ordinary one-way ANOVA with Šidák’s multiple comparisons test was used to compare the means between S1 and S1 P+M at each concentration (ns P>0.05, *P≤0.05, **P≤0.005). (G) Turbidity measurement of FUS LLPS reversal with S1 P+M in TAB-K + ± Mg 2+ . FUS (2 µM) LLPS was initiated, and monitored by turbidity at 395 nm. After 90 min, data collection was paused to add S1 P+M diluted in 10 mM Tris-HCl pH 7.4 supplemented with 50 mM KCl (purple symbols) or supplemented with 50 mM KCl + 3 mM MgCl 2 (green symbols). Data represent mean±SEM (n=3). (H) AUC was calculated from turbidity curves in after RNA was added. Data represent mean±SEM (n=3). Ordinary one-way ANOVA with Šidák’s multiple comparisons test was used to compare the means (ns P>0.05). (I) Representative images of FUS droplets in TAB-K + ± 3 mM MgCl 2 before and after the addition of RNA S1 P+M . FUS (1.8 µM FUS supplemented with 0.2 µM FUS-GFP) droplets were formed in TAB-K + ± 3 mM MgCl 2 for 90 min prior to adding S1 P+M (8 or 32 µM unlabeled RNA). Images were acquired before adding RNA (“pre-RNA”) and 60 min post-RNA addition (“post-RNA”). Scale bar is 5 µm. (J) Representative images of permeabilized HEK 293 cells treated with RNA S1 or S1 P+M after sodium arsenite treatment (0.5 mM, 1 h). Cells were fixed and stained for endogenous FUS (green), TIAR (red), and DAPI (blue). Scale bar is 10 µm. (K) Pearson correlation of cytoplasmic FUS and cytoplasmic TIAR foci from . Bars represent the mean±SEM (n=3). Large squares represent individual trial means (n=3) and small circles represent individual image means (n=75). Ordinary one-way ANOVA with Šidák’s multiple comparisons test was used to compare the indicated means (ns P>0.05, **P≤0.005, ****P<0.0001).

Article Snippet: The Large RNA fraction (S1*10) was concentrated using RNA Clean & Concentrator-5 Kit (Zymo, R1013).

Techniques: Concentration Assay, Staining

Journal: The Journal of Biological Chemistry

Article Title: IRF1 is a context-dependent homeostatic gatekeeper of basal immunity and antiviral readiness

doi: 10.1016/j.jbc.2025.111118

Figure Lengend Snippet: Cytokine profiling and antiviral activity of conditioned media from IRF1 OE cells. A , STAT phosphorylation of HeLa cells (WT) and HeLa IFNAR KO after 30 min of treatment with CM from WT, J AK1 KO (CM-J1), and IFNAR KO (CM-R) IRF1 OE cells, relative to nontreated cells. Quantification of this blot and two more replicates are presented in , A and B . B , IRF1 abundance in HeLa (WT) and HeLa IFNAR KO cells following 6 h of treatment with CM from WT, JAK1 KO, and IFNAR KO cells, relative to nontreated cells. Quantification of this blot is presented in C . C and D , cytometry analysis of IFN-α2 ( C ) and IFN-β ( D ) abundance in cells overexpressing IRF1 (IRF1 OE) or MAVS (MAVS OE) in WT, JAK1 KO, and IFNAR KO cells. WT NT cells were used as a negative control. E , heatmap of the normalized counts of various type I IFNs retrieved from the RNA-seq data. F , CM fractionation and their activity. The blue line represents absorption at 280 nm (mAu), while the red dots represent cell survival after treatment with the given fraction of CM for 4 h, followed by VSV infection for 18 h. Cell viability was determined by crystal violet staining. IFN, interferon; IFNAR, IFN-α receptor; KO, knockout; OE, overexpression; VSV, vesicular stomatitis virus.

Article Snippet: Our hypothesis for the subtle differences between the IRF1 KO and wildtype cells is that the KO exhibits compensatory increased abundance of other IRFs, as shown in , E and F . To evaluate whether abundance of IRF1 relates to abundance of other gene transcripts in multiple different cell types, we analyzed large-scale RNA-seq data from 1206 human cell types using the Human Protein Atlas ( ).

Techniques: Activity Assay, Phospho-proteomics, Cytometry, Negative Control, RNA Sequencing, Fractionation, Infection, Staining, Knock-Out, Over Expression, Virus

Journal: The Journal of Biological Chemistry

Article Title: IRF1 is a context-dependent homeostatic gatekeeper of basal immunity and antiviral readiness

doi: 10.1016/j.jbc.2025.111118

Figure Lengend Snippet: Predicted and observed IRF1 binding in promoter regions of key immune-related genes. A–E , comparison of predicted IRF1-binding affinity and ChIP-seq coverage in promoter regions of genes identified from RNA-seq analysis. The promoter regions include 1000 base pairs upstream and 500 base pairs downstream of the transcription start site. Predicted IRF1-binding affinity is shown in purple and was visualized using inverse log 2 -transformed z-scores (2 ∧ z-score) to represent the relative strength of predicted interactions on a linear scale. Raw ChIP-seq data were reprocessed with a standardized pipeline from GEO datasets ( GSM6928615 , GSM6928616 ) represented in blue , showing IRF1 binding coverage across the promoter regions. Gene annotations are in green . A , gene transcripts showing upregulation in the IRF1 KO, corresponding to cluster 4 from RNA-seq analysis ( C ). B , gene transcripts showing downregulation in the IRF1 KO, corresponding to cluster 1 from RNA-seq analysis ( C ). C , gene transcripts showing upregulation in IRF1 OE, corresponding to cluster 3 from RNA-seq analysis ( A ). D , gene transcripts showing upregulation in both the IRF1 KO and IRF1 OE ( C ). E , analysis of the IFN promoter region, where a subtle ChIP-seq peak aligns with predicted binding sites. Although this peak is less pronounced, it may be functionally significant due to the high sensitivity of IFN receptors, which can initiate an antiviral response even with very low-level of type I IFN. More genes are shown in . GEO, Gene Expression Omnibus; IFN, interferon; IRF1, interferon regulatory factor 1; KO, knockout; OE, overexpression.

Article Snippet: Our hypothesis for the subtle differences between the IRF1 KO and wildtype cells is that the KO exhibits compensatory increased abundance of other IRFs, as shown in , E and F . To evaluate whether abundance of IRF1 relates to abundance of other gene transcripts in multiple different cell types, we analyzed large-scale RNA-seq data from 1206 human cell types using the Human Protein Atlas ( ).

Techniques: Binding Assay, Comparison, ChIP-sequencing, RNA Sequencing, Transformation Assay, Gene Expression, Knock-Out, Over Expression

Journal: The Journal of Biological Chemistry

Article Title: IRF1 is a context-dependent homeostatic gatekeeper of basal immunity and antiviral readiness

doi: 10.1016/j.jbc.2025.111118

Figure Lengend Snippet: Functional analysis of IRF1 regulation of the IFIT2 promoter using prediction, mutagenesis, expression assays, and large-scale transcriptomic correlations. A , normalized RNA-seq expression of IFIT2 in HeLa cells under the indicated conditions: IRF1 KO, IRF1 OE, JAK1 KO + IRF1 OE, IFNAR KO + IRF1 OE, and WT treated with IFN-β for 16 h. B , predicted IRF1 binding affinity across the IFIT2 promoter (chr10:89,300,997–89302495), based on inverse log 2 -transformed z-scores. The analyzed region includes 1000 bp upstream and 500 bp downstream of the transcription start site. Two predicted binding sites near the start codon are highlighted in green . C–F , predicted IRF1 binding sites on the WT IFIT2 promoter ( C ), and three scrambled variants in which one or both predicted IRF1 binding sites were scrambled: Ps 1 ( D ), Ps 2 ( E ), and Ps 1 + 2 ( F ). Binding affinity was recalculated for each sequence and plotted using inverse log 2 -transformed z-scores. G , reporter assay results for the IFIT2 promoter fused with the eUnaG2 gene expressed in WT and IRF1 KO HeLa cells. Cells were transfected with reporter constructs containing the WT promoter or scrambled variants and were either NT, transfected with IRF1 OE, or treated with IFN-β for 12 h. Reporter expression is shown as fold change in fluorescence intensity measured by flow cytometry. Data represent mean ± SD from independent experiments, from which significance was calculated using one-way Anova with post Tukey test. P values of 0.05, and <0.001 are shown as ∗ and ∗∗∗∗. IFIT2, tetratricopeptide repeats 2; IFN, interferon; IFNAR, IFN-α receptor; IRF1, interferon regulatory factor 1; KO, knockout; Ps1, first, higher peak scrambled; Ps2, second (lower) peak scrambled; Ps 1 + 2, both sites scrambled; NT, untreated; OE, overexpression.

Article Snippet: Our hypothesis for the subtle differences between the IRF1 KO and wildtype cells is that the KO exhibits compensatory increased abundance of other IRFs, as shown in , E and F . To evaluate whether abundance of IRF1 relates to abundance of other gene transcripts in multiple different cell types, we analyzed large-scale RNA-seq data from 1206 human cell types using the Human Protein Atlas ( ).

Techniques: Functional Assay, Mutagenesis, Expressing, RNA Sequencing, Binding Assay, Transformation Assay, Sequencing, Reporter Assay, Transfection, Construct, Fluorescence, Flow Cytometry, Knock-Out, Over Expression

Journal: bioRxiv

Article Title: Epilepsy-Associated SCN2A-L1342P Mutation Drives Network Hyperexcitability and Widespread Transcriptomic Changes in Human Cortical Organoids

doi: 10.1101/2025.08.18.670956

Figure Lengend Snippet: (A) Bulk RNA-seq experimental conditions. Control organoids (n = 6 organoids, three differentiations) and L1342P organoids (n = 6 organoids, two differentiations) were sequenced using the AVITI system. (B) Hierarchical clustering of cortical organoid sample replicates. Darker blue indicates higher similarity. (C) Principal Component Analysis (PCA) plot illustrating the clustering of replicates based on their variance in two dimensions. (D) DEG Heatmap showing the top 50 down (blue) and upregulated (red) genes across conditions. (E) Volcano plot highlighting significantly differentially expressed genes (DEGs, FDR < 0.05) in Nav1.2-L1342P hiPSC-derived cortical organoids compared to controls. Notable downregulated genes include SCN2A and potassium channel-related genes. In contrast, upregulated genes include: DLX2 (Distal-Less Homeobox 2), H2AX (histone family member X), PAX6 (Paired Box 6), and ASCL1 (Achaete-scute family bHLH transcription factor 1), which are important in neurodevelopment, neuronal function, and cellular function. (F) Gene Ontology (GO) analysis of biological processes reveals changes in synapse organization, microtubule-cytoskeleton arrangements, glutamate receptor signaling, and forebrain development (top). (G) GO molecular function analysis reveals enrichment in ion channel activity and alterations in the glutamatergic pathway. (H) GO cellular component identifies synaptic pathway alterations in the Nav1.2-L1342P cortical organoids. (I) Network analysis reveals numerous globally differentially expressed genes (DEGs) involved in synaptic signal regulation (magenta), calcium transport (dark green), synapse organization (orange), and forebrain development (lime). The colored edges connecting genes represent functional relationships or interactions within each cluster, highlighting how these DEGs collectively contribute to altered neuronal physiology. (J) REACTOME pathway analysis reveals disruptions in cell division, metabolism, AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor trafficking, and neuronal synapses. (K) KEGG pathway enrichment analysis reveals alterations in MAPK and cAMP signaling pathways, as well as glutamatergic synapses. The circle size is represented by the number of DEGs highly ranked by p.adjust values.

Article Snippet: To this end, we extracted total RNA from the cortical organoids using the NucleoSpin miRNA Kit for the Isolation of Small and large RNA (Macherey-Nagel, Catalog No. 740971.50) according to the manufacturer’s instructions.

Techniques: RNA Sequencing, Control, Derivative Assay, Cell Function Assay, Activity Assay, Functional Assay, Protein-Protein interactions