Journal: Science Advances

Article Title: Accessory microRNA byproducts expand RNA interference via microprocessor-mediated cleavage activation

doi: 10.1126/sciadv.adz8331

Figure Lengend Snippet: ( A ) RNA structure prediction of CL3 construct. Each component is color-coded and labeled. ( B ) Volcano plot showing changes in short ncRNA from G34 patient-derived GBM stem-like cells expressing GFP-CL3 transgene versus GFP-only (CTRL) transgene. Results are based on three biological replicates per group. Gray dots represent P values > 0.05. ( C ) Agarose gel (1%) Northern blot of whole cell RNA obtained from HEK293 cells expressing CL3 and CTRL, same as described in (B). Cartoon shows the target of each tested probe within the transgene sequence. Stars denote the primary cluster sequence. Arrowheads denote cleaved flanking regions of the primary transcript. ( D ) RT-qPCR from RNA of G34 cells expressing CL3 grown in normal medium or in the presence of actinomycin D (10 μg/ml) for 6 hours. Color-coded RNA structures represent the predicted folding of the 3′ and 5′ flanks, respectively. Bars represent mean values + SD; n = 3 biological replicates. Each comparison was analyzed with Student’s t test, two tails. FC, fold change; RQ, relative quantification. ( E ) RT-qPCR of G34 cells expressing a modified CL3 sequence where the 5′ flank is replaced with a Tough Decoy (TuD), whose predicted structure is shown above. Cells were grown in normal medium or in the presence of actinomycin D (10 μg/ml) for 6 hours. Bars represent mean values + SD; n = 3 biological replicates. Each comparison was analyzed with Student’s t test, two tails. ( F ) Rationale for the design of a new microRNA-based platform. **** P < 0.0001; ns, nonsignificant.

Article Snippet: A total of 10 ng of DNA and RNA was added per PCR reaction and amplified using the Luna Universal Probe One-Step RT-qPCR Kit (catalog no. E3006L, New England Biolabs) following the manufacturer’s protocol.

Techniques: RNA Structure Prediction, Construct, Labeling, Derivative Assay, Expressing, Agarose Gel Electrophoresis, Northern Blot, Sequencing, Quantitative RT-PCR, Comparison, Quantitative Proteomics, Modification

Journal: Science Advances

Article Title: Accessory microRNA byproducts expand RNA interference via microprocessor-mediated cleavage activation

doi: 10.1126/sciadv.adz8331

Figure Lengend Snippet: ( A ) Schematics and corresponding predicted RNA folding of engineered microRNA sequences. Each component is labeled and color-coded accordingly. The black arrow represents the site of Drosha cleaved along the miR-7 stem sequence. ( B ) microRNA expression in HEK293 cells infected with different constructs. Bars represent mean values + SD; n = 3 biological replicates. Statistical analysis was performed by two-way ANOVA compared with SCR with Dunnett’s multiple comparisons test. ( C ) Polyacrylamide gel (4 to 20%) Northern blot from whole cell RNA of HEK293 cells transfected with different constructs. ( D ) Rationale for the design of a new microRNA-based platform. Mi-dAp50, microRNA-driven aptamer anti-p50. ( E ) PCR analysis of Mi-dAp50 in samples after RIP by p50 versus immunoglobulin G bait. Bars represent mean values ± SD; n = 3 biological replicates. Statistical analyses were performed by Student’s t test, two tails. ( F ) RT-qPCR showing gene expression level of known NF-κB target genes. RNA was isolated from HEK293 cells expressing different constructs. Bars represent mean values + SD; n = 3 biological replicates. Each comparison was analyzed with Student’s t test, two tails. ( G ) Luminescence detection in HEK293 expressing NF-κB responsive luciferase reporter genes and transfected with different clusters and specific, in presence or absence of TNF-α (10 ng/ml). Parallel experiments were done in Drosha WT and Drosha KO models. Bars represent mean values ± SD; n = 3 biological replicates. Statistical analysis was performed by Student’s t test, two tails. RLU, relative luminescence unit. ( H ) ChIP-PCR of p50 precipitated DNA from HEK293 cells expressing different constructs. The drawing clarifies IKBα as a known NF-κB responsive gene, while RLP30 is used as a negative control. Bars represent mean values ± SD; n = 3 biological replicates. Statistical analysis was performed by one-way ANOVA. * P < 0.05; ** P = < 0.01; *** P = < 0.001; **** P < 0.0001; ns, nonsignificant. TSS, transcription start site.

Article Snippet: A total of 10 ng of DNA and RNA was added per PCR reaction and amplified using the Luna Universal Probe One-Step RT-qPCR Kit (catalog no. E3006L, New England Biolabs) following the manufacturer’s protocol.

Techniques: Labeling, Sequencing, Expressing, Infection, Construct, Northern Blot, Transfection, Quantitative RT-PCR, Gene Expression, Isolation, Comparison, Luciferase, Negative Control

Journal: Science Advances

Article Title: Accessory microRNA byproducts expand RNA interference via microprocessor-mediated cleavage activation

doi: 10.1126/sciadv.adz8331

Figure Lengend Snippet: ( A ) Fluorescence microscopy of T98G GBM cells expressing different RNA cluster constructs and infected with oHSV-1 [multiplicity of infection (MOI) = 0.5], acquired 12 hours after infection. Scale bar, 100 μm. ( B ) Fluorescence-activated cell sorting analysis for detection of GFP (oHSV-1 marker) in G34 cells expressing different RNA cluster constructs. ( C ) Quantification of cell viability with alamarBlue, 24 hours after oHSV-1 infection of T98G cells (MOI = 0.5), expressing different RNA constructs. Bars represent mean ± SD; n = 4 biological replicates. Statistical analysis by one-way ANOVA. ( D ) RT-qPCR quantification of oHSV-1 gene expression at different time points in G34 cells infected at MOI = 0.5. Bars represent mean values ± SD; n = 3 biological replicates. Statistical analysis by one-way ANOVA. ( E ) Representative images of plaque assay in Vero 2.2 cells incubated with an equal volume of conditioned supernatant from previously oHSV-1–infected (MOI = 0.5) G34 cells expressing different constructs. Plaques are visualized by GFP. Scale bar, 500 μm. ( F ) Kaplan-Meier survival curve of female athymic nu/nu mice intracranially implanted with 10,000 G34 cells stably expressing the different microRNAs constructs; n = 6. Log rank test, corrected by Bonferroni analysis. Arrowed lines represent different survival gains by oHSV-1. Graphic created in BioRender. Mazzetti, D. (2025) https://BioRender.com/ferhtf3 . ( G ) Fluorescence microscopy images of cryosectioned brain slices obtained 9 days after oHSV-1 infection. Scale bars, 1000 μm (full brain); 25 μm (inset). ( H ) RT-qPCR analysis of oHSV-1 gene expression from tumor tissues referred to (G). Bars represent mean values + SD; n = 3 biological replicates. Each comparison was analyzed with Student’s t test, two tails. ( I ) Copy number quantification oHSV-1 genome recovered from tumor tissue related to (G) and (H). Bars represent mean values + SD; n = 3 biological replicates. Each comparison was analyzed with Student’s t test, two tails. * P < 0.05; ** P = < 0.01; **** P < 0.0001; ns, nonsignificant. FSC-A, forward scatter area.

Article Snippet: A total of 10 ng of DNA and RNA was added per PCR reaction and amplified using the Luna Universal Probe One-Step RT-qPCR Kit (catalog no. E3006L, New England Biolabs) following the manufacturer’s protocol.

Techniques: Fluorescence, Microscopy, Expressing, Construct, Infection, FACS, Marker, Quantitative RT-PCR, Gene Expression, Plaque Assay, Incubation, Stable Transfection, Comparison