rna motif plasmid  (Addgene inc)

Journal: bioRxiv

Article Title: UCHL3 regulates subgenomic flaviviral RNA condensates to promote virus propagation

doi: 10.1101/2025.09.05.674517

Figure Lengend Snippet: (A) Co-immunoprecipitation analysis of Flag-HA-UCHL3 from 293 cells transfected with either empty vector control (pcDNA3.1) or Flag-HA-UCHL3 construct and infected with ZIKV or mock-infected. Input lysates and immunoprecipitated (IP) fractions were analysed by immunoblotting for viral non-structural proteins NS5, NS3, and NS4A, viral structural protein prM, and Flag-HA-UCHL3. (B) Schematic representation of the biotinylated sfRNA-interactome capture strategy. The engineered sfRNA construct contains flanking BoxB stem-loop structures that specifically recruit the BirA-λN fusion protein (BASU). Upon biotin supplementation, BASU biotinylates proteins in close proximity to sfRNA, enabling streptavidin-mediated affinity purification of sfRNA-associated ribonucleoprotein complexes. (C) Silver staining analysis of sfRNA-interacting protein complexes. Total protein profiles from streptavidin pulldowns using control RNA motif, ZIKV sfRNA, or DENV sfRNA constructs in the presence or absence of ZIKV infection for infection-dependent recruitment of cellular factors to sfRNA assemblies. (D) Streptavidin-affinity capture followed by immunoblot analysis showing selective enrichment of UCHL3 and G3BP1 in the presence of ZIKV or DENV sfRNA constructs. Cell lysates and streptavidin pulldown (SA-Pulldown) fractions show specific association of these proteins with sfRNA-containing complexes. BASU (HA-tagged) serves as an internal control for the capture system. (E-F) Quantitative RT-PCR analysis of (E) NS5 genomic RNA and (F) sfRNA levels in cells transfected with either pcDNA3.1 empty vector or Flag-HA-UCHL3 at 24 and 48 hours post-ZIKV infection. Ectopic UCHL3 expression increases both viral genomic and subgenomic RNA species compared to vector controls. Data represent mean ± SD from triplicate experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The resulting amplicon and a control RNA motif plasmid cloning backbone were digested with the BsmBI-v2 restriction enzyme (NEB) for 1h at 55°C.

Techniques: Immunoprecipitation, Transfection, Plasmid Preparation, Control, Construct, Infection, Western Blot, Affinity Purification, Silver Staining, Quantitative RT-PCR, Expressing

Journal: bioRxiv

Article Title: Dual function of ERH in primary miRNA biogenesis

doi: 10.1101/2025.09.23.678008

Figure Lengend Snippet: A . Scaled scheme of the human DGCR8 mRNA, with individual exons depicted as numbered boxes. The ORF is marked in dark grey. B . Expression of pre-miR-3618 and miR-1306 as quantified by RT-PCR. Individual datapoints are illustrated as colored circles and triangles depending on the sgRNA (n=3+3). C . PCR-quantified expression of depicted miRNAs and mRNAs in control and miR-3618 KO cells. D , E . Expression of DGCR8 and DROSHA mRNAs in SAFB1/2 and ERH KO cells transduced with different sgRNAs, and the corresponding western blot analysis for protein expression and a quantification thereof. F . Expression of miR-1306 and DGCR8 RNA as well as protein levels in SAFB1/2 KO cells reconstituted with the depicted SAFB2 variants. Numbers indicate p-values.

Article Snippet: BoxB constructs for the mutated variants of miR-15a and -181b were generated based on the RNA motif plasmid (Addgene #107253, kindly provided by Paul Khavari) of the RaPID system , and were expressed with the BoxB-miRNA cassette placed in the 3’-UTR of a cDNA encoding a intracellular tail-deletion mutant of the cell surface marker CD8.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Transduction, Western Blot

Journal: bioRxiv

Article Title: Dual function of ERH in primary miRNA biogenesis

doi: 10.1101/2025.09.23.678008

Figure Lengend Snippet: A . Model for the temporal sequence of cluster assistance, beginning with Microprocessor recruitment to the helper, followed by helper processing and subsequent transfer of DROSHA/DGCR8 to the recipient. B . Predicted structure of the cluster assistance-recipient pri-miR-181b and its seed mutant used for the tethering experiments. C . Control experiments for the Microprocessor tethering as in , indicating no miRNA reporter repression in the absence on any BoxB aptamer sites in the substrate RNA. D . Corresponding experiment to , using tethering of λN-DGCR8 to miR-15a mut and its respective reporter as a readout. Histogram overlays and the bar graph depict changes in dsRed fluorescence in consequence of SAFB1/2 and ERH knockout. Data points are shown as circles and triangles for the individual sgRNAs (n=3+3). Numbers indicate p-values.

Article Snippet: BoxB constructs for the mutated variants of miR-15a and -181b were generated based on the RNA motif plasmid (Addgene #107253, kindly provided by Paul Khavari) of the RaPID system , and were expressed with the BoxB-miRNA cassette placed in the 3’-UTR of a cDNA encoding a intracellular tail-deletion mutant of the cell surface marker CD8.

Techniques: Sequencing, Mutagenesis, Control, Fluorescence, Knock-Out