clontech matchmaker gold yeast  (TaKaRa)

Journal: The Journal of cell biology

Article Title: DUX4-induced HSATII RNA accumulation drives protein aggregation impacting RNA processing pathways

doi: 10.1083/jcb.202501129

Figure Lengend Snippet: ( A ) Immunofluorescence of EU-RNA aggregates (gray) in iDUX4 cells that were left untreated (−DOX) or pulsed for 4-hours using 1ug/mL doxycycline (+DOX) and then incubated with 0.1mM 5-ethynyluridine (+EU) for 16-hours prior to washout and fixed at 24-hour time points (24–96 hours). Control cells were uninduced (−DOX) iDUX4 cells incubated with EU and fixed at either 24 hours or 96 hours or without EU and fixed at 24 hours. Scale bar = 20μm. Images are representative of two independent experimental replicates performed. ( B ) Percent of cells with intranuclear EU-RNA aggregates in −DOX or +DOX iDUX4 cells labeled with EU for 16-hours at 24-hour time points. EU-RNA aggregates are present within +DOX iDUX4 cells from 24-hour to 72-hour time points. N=2 experimental replicates. Minimum 200 nuclei per experiment. Data represent means ± standard deviation (SD). Statistical differences between groups were analyzed employing one-way ANOVA Dunnett’s multiple comparisons test between each group and a control (−DOX+EU 24-hour time point). ( C ) Enrichment of RPL27 or HSATII RNA in isolated EU-RNA from −DOX or +DOX iDUX4 cells incubated with EU for 16-hours or +DOX iDUX4 cells with no EU treatment, all harvested at the 48-hour time point. Housekeeping gene RPL27 is used as a control. N=3. A total of four independent experimental replicates were performed. Data represent means ± SD. ( D ) Quantitative RT-PCR of HSATII expression (2 −DCt ) in −DOX or +DOX iDUX4 cells treated with EU for 16-hours or +DOX iDUX4 cells with no EU treatment, all harvested at the 48-hour time point. EU-treatment does not impact HSATII RNA expression. N=3. A total of four independent experimental replicates were performed. Data represent means ± SD. Statistical differences between groups were analyzed employing one-way ANOVA Dunnett’s multiple comparisons test between each group and a control (−DOX+EU). ( E ) Enrichment of RPL27 or HSATII RNA in isolated EU-RNA from −DOX or +DOX iDUX4 cells incubated with EU for 16-hours and harvested at 24-hour time points from 48-hours to 96-hours. Housekeeping gene RPL27 is used as a control. N=3. A total of three independent experimental replicates were performed. Data represent means ± SD. ( F ) Combined immunofluorescence and HSATII RNA-fluorescence in situ hybridization (RNA-FISH) of HSATII RNA (green) and EU-RNA (magenta) in +DOX+EU iDUX4 cells fixed at 48-hour time point. Scale bar = 20μm. Images are representative of three independent experimental replicates performed. ( G ) Percent of cells with intranuclear EU-RNA aggregates only (“EU”), HSATII RNA aggregates only (“HSATII”) or both EU-RNA/ HSATII RNA aggregates (“both”) in +DOX+EU iDUX4 cells fixed at the 48-hour time point. N=3 experimental replicates. Minimum 200 nuclei per experiment. Data represent means ± SD. ( H ) Combined immunofluorescence and HSATII RNA-FISH of HSATII RNA (green) and EU-RNA (magenta) in control depleted (“CTRL KD”) or HSATII RNA-depleted (“HSATII KD”) +DOX+EU iDUX4 cells fixed at 48-hour time point. Scale bar = 20μm. Images are representative of two independent experimental replicates performed. ( I ) Percent of cells with intranuclear RNA aggregates: EU only, HSATII only or both in CTRL KD or HSATII KD +DOX+EU iDUX4 cells fixed at the 48-hour time point. N=2 experimental replicates. Minimum 200 nuclei per experiment. Data represent means ± SD. Statistical differences between groups were analyzed employing two-way ANOVA Šídák’s multiple comparisons test between CTRL KD and HSATII KD within each group (EU, HSATII, both). Fig 1G and 1L : The frequency of cells containing RNA aggregates exhibits some variability across the population. This variability could be due to the robustness and induction efficiency of doxycycline treatment. ( J ) Combined immunofluorescence (IF) of EU-RNA (green) and Fibrillarin (magenta) in −DOX+EU or +DOX+EU iDUX4 cells fixed at 48-hour time point or combined immunofluorescence and HSATII RNA-FISH of HSATII RNA (green) and fibrillarin (magenta) in −DOX and +DOX iDUX4 cells fixed at 24-hours. Scale bar = 20μm. Arrows indicate nuclei with disrupted nucleolar staining. Images are representative of two independent experimental replicates performed. ( K ) Immunofluorescence (IF) of EU-RNA (green) and nucleophosmin-1 (NPM1) (magenta) in −DOX+EU or +DOX+EU iDUX4 cells fixed at 48-hour time point or combined immunofluorescence and HSATII RNA-FISH of HSATII RNA (green) and NPM1 (magenta) in −DOX and +DOX iDUX4 cells fixed at 24-hours. Scale bar = 20μm. Arrows indicate nuclei with disrupted nucleolar staining. Images are representative of two independent experimental replicates performed. ( L ) Percent of cells with nucleolar disruption present in cells with intranuclear RNA aggregates. N=2 experimental replicates. Minimum 300 nuclei per experiment.

Article Snippet: Hybridization was performed at 37°C for 4 hours shaking at 800rpm using biotin-conjugated probes (HSATII probe: 5′-ATTCCATTCAGATTCCATTCGATC-3BioTEG-3’, Control probe: 5’-GTCCCGTTAGCTCAGGTGGTAGAGCAC-3BioTEG-3’ or 5’-TGCTGATGAAGCAGAACAAC-3BioTEG-3’, MALAT1 probe sequences were taken from ( Miao et al., 2022 ): 5’-AGATATTGTGCTGTTACCTC-3BioTEG-3’, 5’-TTCTCTGGCCCTTCGCATAC-3BioTEG-3’, 5’-CCCAAGATTGCCCCAACACT-3BioTEG-3’, 5’-CACCCTCTAAGAGACATTCA-3BioTEG-3’, 5’-CCTCAGTTACACATCCAAAC-3BioTEG-3’, 5’-GTACATTTTGCCCTTAGCTT-3BioTEG-3’, 5’-CCAGTGGCCCACTCTGATCT-3BioTEG-3’) in hybridization buffer (750 mM NaCl, 1% SDS, 50 mM Tris-HCl, pH 7.0, 1 mM EDTA, 15% formamide (Sigma-Aldrich) and supplemented with fresh 0.5 mM AEBSF (ThermoFisher Scientific, cat# BP635–500), Pierce Protease Inhibitors EDTA-free ( PIA32955 ) and Pierce Phosphatase Inhibitors ( PIA32957 ) and RNase inhibitor (TaKaRa, cat# 2313A)).

Techniques: Immunofluorescence, Incubation, Control, Labeling, Standard Deviation, Isolation, Quantitative RT-PCR, Expressing, RNA Expression, Fluorescence, In Situ Hybridization, Staining, Disruption

Journal: Science Advances

Article Title: OsRNS4-mediated cross-kingdom RNA interference is inhibited by Ustilaginoidea virens effector SCRE2 to promote infection in rice

doi: 10.1126/sciadv.adx7385

Figure Lengend Snippet: ( A ) Yeast two-hybrid assay to show the interaction of SCRE2nsp with OsRNS4. The pGADT7 (AD) and pGBKT7 (BD) constructs were cotransformed into yeast GOLD strain. The transformants were cultured on the dropout medium plates. AD-T and BD-53, a positive control; AD-T and BD-λ, a negative control. OsRNS6, an OsRNS4 homolog in rice RNase T2 family. SCRE1, another U. virens effector. ( B ) Co-IP assay showing a specific interaction between SCRE2 and OsRNS4 in rice protoplasts. SCRE2-FLAG was expressed alone or coexpressed with OsRNS4-HA or OsRNS6-HA in rice protoplasts. Total proteins from transfected protoplasts were incubated with anti-FLAG M2 affinity beads. The inputs and immunoprecipitates were analyzed via immunoblotting with anti-HA (α-HA) and anti-FLAG (α-FLAG) antibodies. IP, immunoprecipitation; WB, Western blot. ( C ) In vitro pull-down assay to detect direct interaction between SCRE2 and OsRNS4. Purified GST- and His-tagged proteins were coincubated with glutathione agarose beads. The input and pull-down proteins were detected with anti-His (α-His) and anti-GST (α-GST) antibodies.

Article Snippet: Yeast two-hybrid assay was performed using the Clontech Matchmaker Gold Yeast Two-Hybrid System following the manufacturer’s instructions (Clontech).

Techniques: Y2H Assay, Construct, Cell Culture, Positive Control, Negative Control, Co-Immunoprecipitation Assay, Transfection, Incubation, Western Blot, Immunoprecipitation, In Vitro, Pull Down Assay, Purification