Journal: bioRxiv

Article Title: SAMHD1 controls innate immunity by regulating condensation of immunogenic self RNA

doi: 10.1101/2022.07.12.499661

Figure Lengend Snippet: ( A ) Schematic illustration of the human SAMHD1 protein and its functional domains (SAM domain, aa45-110 and catalytic HD domain, aa164-316). Investigated mutations (H167Y, Q548X, and T592A) are highlighted. ( B ) Coomassie Blue-stained SDS-PAGE of purified GST-SAMHD1. ( C ) Size exclusion chromatography profiles of GST-SAMHD1 alone (red) or in the presence of 200 µM dGTP (blue). ( D ) Western blot of wild type (GST-SAMHD1-WT) and phosphorylation-deficient SAMHD1 (GST-SAMHD1-T592A) after treatment with recombinant cyclin A2/cyclin-dependent kinase (CDK) 1, immunostained with antibody against SAMHD1 or phospho-SAMHD1 (T592). ( E ) Last three lanes of each panel: RNase activity of phosphorylated wild type SAMHD1 (WT p592) and phosphorylation-deficient SAMHD1 (T592A) after treatment with recombinant cyclin A2/CDK1, and wild type SAMHD1 in the presence of 200 µM dGTP (WT + 200 µM dGTP) using the following substrates: total RNA, mRNA and RNA:DNA hybrid with 5’ diphosphate on RNA (5’ pp RNA:DNA). Buffer, negative control. Benzo, benzonase, positive control. First lane, ssRNA ladder; last lane, dsRNA ladder. ( F ) RNase activity of wild type SAMHD1 without or in the presence of 200 µM dGTP using total RNA as substrate. ( G ) RNase activity of wild type SAMHD1 without or in the presence of the indicated divalent cations (either 2 mM Mg 2+ , 200 pM Zn 2+ , 2 mM Ca 2+ and 200 pM Zn 2+ , 2 mM Mg 2+ and 200 pM Zn 2+ or a modified buffer containing 3 mM NaCl, 20 mM KCl, 10 mM Tris-HCl (pH 7.5) and 2 mM Mg 2+ , 200 pM Zn 2+ and 200 µM Ca 2+ ) using total RNA as substrate. ( H ) RNase activity of wild type (WT) and mutant (Q548X, H167Y, T592A) SAMHD1 using the following substrates: dsRNA carrying 5’ triphosphate (5’ppp dsRNA), primary micro-RNA (miRNA), RNA:DNA hybrid with 5’ triphosphate on RNA (5’ ppp RNA:DNA), small interfering RNA (siRNA) and yeast 70S ribosome. Buffer, negative control. Benzo, benzonase, positive control. First lane, ssRNA ladder; last lane, dsRNA ladder.

Article Snippet: Mouse brain polyA+ RNA (ZYAGEN, MR-201_MR) and human brain microRNA (ZYAGEN, HR-201-SR) were purchased from Amsbio.

Techniques: Functional Assay, Staining, SDS Page, Purification, Size-exclusion Chromatography, Western Blot, Recombinant, Activity Assay, Negative Control, Positive Control, Modification, Mutagenesis, Small Interfering RNA

Journal: bioRxiv

Article Title: SAMHD1 controls innate immunity by regulating condensation of immunogenic self RNA

doi: 10.1101/2022.07.12.499661

Figure Lengend Snippet: ( A ) mRNA expression of condensate-specific constituent proteins in AGS patient cells and wild type controls. ( B , C ) Mean fluorescence intensity (MFI) of G3BP1 in stress granules relative to cytoplasm (n = 94-135 cells, B ) and of SC35 in nuclear speckles (n = 222-559 cells, C ). Box plots: center line, median; box, interquartile range; whiskers, 1.5x interquartile range; dots, outliers. Kruskal-Wallis test with Dunn’s multiple comparisons test. **, p<0.01; ***, p<0.001. Data are representative of three independent experiments. ( D - G ) Images and quantification of membraneless compartments in HeLa cells transfected with control-shRNA (Co) or SAMHD1-shRNA (SAMHD1). White dotted lines mark the cellular boundary, and yellow dotted lines denote the nuclear outline. Scale bars, 5 μm. G3BP1, n = 41-89 cells; SC35, n = 61 cells; FIB, n = 56-60 cells; DDX21, n = 41-89 cells. Box plots: center line, median; box, interquartile range; whiskers, 1.5x interquartile range; dots, outliers. t-test. **, p<0.01; ***, p<0.001. Data are representative of three independent experiments.

Article Snippet: Mouse brain polyA+ RNA (ZYAGEN, MR-201_MR) and human brain microRNA (ZYAGEN, HR-201-SR) were purchased from Amsbio.

Techniques: Expressing, Fluorescence, Transfection, shRNA

Journal: bioRxiv

Article Title: SAMHD1 controls innate immunity by regulating condensation of immunogenic self RNA

doi: 10.1101/2022.07.12.499661

Figure Lengend Snippet: ( A ) Representative images of stress granule-like condensates induced by addition of AF-488-labeled SNAP-G3BP1 to HeLa cell lysates to which either Cy5-labeled ssRNA or dsRNA was added, along with color-coded intensity images of RNA and G3BP1 in stress granule-like condensates. Scale bars, 5 µm. ( B ) Quantification of G3BP1 fraction in stress granule-like condensates (cumulative G3BP1 intensity in condensates per total G3BP1). n =1000-1450 condensates. Mean ± SD. ( C ) Representative images of minimal G3BP1 condensates induced by adding poly(A)-RNA to AF 488-labeled SNAP-G3BP1 in physiological buffer to which ssRNA or dsRNA was added. Scale bars, 5 µm. ( D ) Quantification of G3BP1 fraction within G3BP1 condensates in presence or absence of ssRNA or dsRNA. n = 30-45 field of views. Box plots: center line, median; box, interquartile range; whiskers, 1.5x interquartile range; dots, outliers. Kruskal-Wallis test with Dunn’s multiple comparisons test. **, p<0.01; ***, p<0.001. ( E ) Representative images of stress granule-like condensates induced by addition of AF 488-labeled SNAP-G3BP1 to HeLa cell lysates to which either Cy5-labeled ssRNA (45 nt) or dsRNA (45 bp) was added, along with color-coded intensity images of G3BP1. Scale bars, 5 µm. ( F ) Quantification of proportion of stress granule-like condensates containing RNA aggregates. n = 25 field of views. Box plots: center line, median; box, interquartile range; whiskers, 1.5x interquartile range; dots, outliers. Mann-Whitney U test. ***, p<0.001. ( G ) Representative images of aggregates in stress granule-like condensates build after mixing AF 488-labeled SNAP-G3BP1 and dsRNA of increasing size (Cy5-labeled 45 bp dsRNA, Rhodamine-labeled low molecular weight or high molecular weight poly(I:C)) in HeLa cell lysate along with color-coded RNA intensity. Scale bars, 5 µm.

Article Snippet: Mouse brain polyA+ RNA (ZYAGEN, MR-201_MR) and human brain microRNA (ZYAGEN, HR-201-SR) were purchased from Amsbio.

Techniques: Labeling, MANN-WHITNEY, Molecular Weight

Journal: bioRxiv

Article Title: SAMHD1 controls innate immunity by regulating condensation of immunogenic self RNA

doi: 10.1101/2022.07.12.499661

Figure Lengend Snippet: ( A ) Western blot of wild type (WT) and AGS fibroblasts treated with control-siRNA (Co) or siRNA against cGAS, MDA5, or RIG-I, immunostained with the indicated antibodies. ( B ) Expression of IFN-stimulated genes in AGS cells treated with indicated siRNA, relative to wild type cells treated with control-siRNA. Mean ± SEM of three independent experiments run in triplicate technical replicates. One-way ANOVA with Dunnett’s multiple comparisons test, co-siRNA vs. sensor-siRNA. *, p<0.05. ( C , D ) IP-10 secretion of wild type HeLa cells (WT) or HeLa cells with knockout of MDA5 (MDA5 -/- ) or RIG-I (RIG-I -/- ) or with double knockout of MDA5 and RIG-I (MDA5 -/- /RIG-I -/- ; dKO) treated with control-siRNA or SAMHD1-siRNA after stimulation with 5’ppp dsRNA ( C ) and EMCV total RNA ( D ). Mean ± SEM of two independent experiments with four technical replicates. Two-way ANOVA with Sidak’s multiple comparisons test. ***, p<0.001. ( E ) Representative Western blot of GFP-DYRK3 expressing HeLa cells without or with 1 h doxycycline treatment showing induction of p-STAT1 and MX1 after 3 h of recovery. ( F ) Expression of CXCL10 mRNA in GFP-DYRK3 expressing HeLa cells either untreated or treated with doxycycline for 1 h, followed by 2 h or 3 h recovery. Mean ± SEM of triplicate technical replicates, representative of three independent experiments. One-way ANOVA with Dunnett’s multiple comparisons test, doxycycline untreated vs. treated, ***, p<0.001. ( G ) Model: in normal cells, the RNase activity of SAMHD1 controls cellular levels of ssRNA, which together with RNA-binding proteins, are required for condensate formation by phase separation. In SAMHD1 deficiency, unmetabolized ssRNA accumulates, impeding formation of condensates which sequester dsRNA. This leads to release of dsRNA from condensates with subsequent activation of RLR-dependent type I IFN signalling.

Article Snippet: Mouse brain polyA+ RNA (ZYAGEN, MR-201_MR) and human brain microRNA (ZYAGEN, HR-201-SR) were purchased from Amsbio.

Techniques: Western Blot, Expressing, Knock-Out, Double Knockout, Activity Assay, RNA Binding Assay, Activation Assay

Journal: eLife

Article Title: Transcriptional profiling of Hutchinson-Gilford Progeria syndrome fibroblasts reveals deficits in mesenchymal stem cell commitment to differentiation related to early events in endochondral ossification

doi: 10.7554/eLife.81290

Figure Lengend Snippet: RNA-seq datasets from progeria patients used in this study.

Article Snippet: AG03199 , 10 yr , F , White , polyA (Illumina SureSelect Strand Specific RNA library Prep) , Arufe , GSE113648.

Techniques: Sample Prep

Journal: eLife

Article Title: Transcriptional profiling of Hutchinson-Gilford Progeria syndrome fibroblasts reveals deficits in mesenchymal stem cell commitment to differentiation related to early events in endochondral ossification

doi: 10.7554/eLife.81290

Figure Lengend Snippet: RNA-seq datasets from adult controls used in this study.

Article Snippet: AG03199 , 10 yr , F , White , polyA (Illumina SureSelect Strand Specific RNA library Prep) , Arufe , GSE113648.

Techniques:

Journal: eLife

Article Title: Transcriptional profiling of Hutchinson-Gilford Progeria syndrome fibroblasts reveals deficits in mesenchymal stem cell commitment to differentiation related to early events in endochondral ossification

doi: 10.7554/eLife.81290

Figure Lengend Snippet: RNA-seq datasets from children controls used in this study.

Article Snippet: AG03199 , 10 yr , F , White , polyA (Illumina SureSelect Strand Specific RNA library Prep) , Arufe , GSE113648.

Techniques: Sample Prep

Journal: Wellcome Open Research

Article Title: The genome sequence of the sallow kitten, Furcula furcula (Clerck, 1759)

doi: 10.12688/wellcomeopenres.18112.1

Figure Lengend Snippet: Genome data for Furcula furcula , ilFurFurc1.1.

Article Snippet: PolyA RNA-Seq Illumina , ERR9434990.

Techniques:

Journal: Wellcome Open Research

Article Title: The genome sequence of common ivy, Hedera helix L., 1753

doi: 10.12688/wellcomeopenres.19662.1

Figure Lengend Snippet: Genome data for Hedera helix , drHedHeli1.2.

Article Snippet: PolyA RNA-Seq Illumina , ERR9435019 , .

Techniques: DNA Sequencing