Journal: Molecular Cell
Article Title: RNA Helicase DDX1 Converts RNA G-Quadruplex Structures into R-Loops to Promote IgH Class Switch Recombination
Figure Lengend Snippet: DDX1 Binds to G4 Structures in Intronic Switch RNAs (A) RNA oligonucleotides consisting of 4 tandem Sμ repeats (Sμ4G) or a G-to-C mutant (Sμ4Gmut). (B and C) RNA pull-down assays with protein extracts from (B) AID FLAG-HA or (C) AID KO CH12 cells, CIT stimulated for 48 hr. Western blots were analyzed for DDX1 and AID (FLAG tag) and RNA recovered from beads measured by dot blot. Representative results from at least 3 independent pull-downs. (D) Native electrophoretic mobility shift assays (EMSA) with 32 P-labeled Sμ4G and Sμ4Gmut RNA oligonucleotides and rDDX1 (WT) or rDDX1-K52A (ATPase mutant) proteins (1, 2, or 4 μg). Representative results from at least 3 independent assays. (E–H) CH12 cells were transfected with a pcDNA3 vector expressing GFP or N-terminal GFP-tagged human DDX1-K52A cDNA (GFP::DDX1-K52A), and cultured in UNS or CIT-stimulated conditions. (E) Percentage of GFP + cells 24 hr and 40 hr after transfection measured by flow cytometry (n = 4, mean ± SD). (F) Western blot of GFP + , fluorescence-activated cell sorted cells for DDX1 and Tubulin loading control (24 hr after transfection, 2 replicates). (G) Quantification of CSR in GFP – and GFP + -gated cell populations (40 hr after transfection; n = 4, mean ± SD). (H) DIP analyses with S9.6 antibody (IP) or no antibody control (–), 24 hr after transfection in CIT-stimulated conditions using Sα region probe 9. Values were normalized to probe 2 in each sample and probe 9 in shCtrl CIT cells in each experiment (n = 3, mean ± SD). See also Figure S4 .
Article Snippet: Libraries were prepared from 100 ng RNA using the NEBNext Ultra Directional RNA Library Prep kit and NEBnext Multiplex Oligos (Index Primers Set 2) for Illumina according to manufacturer’s instructions (NEB).
Techniques: Mutagenesis, Hemagglutination Assay, Gene Knockout, Western Blot, FLAG-tag, Dot Blot, Electrophoretic Mobility Shift Assay, Labeling, Transfection, Plasmid Preparation, Expressing, Cell Culture, Flow Cytometry, Cytometry, Fluorescence