Fig. 5: ADAR2 binding to dsRNA that involves the Py-tract blocks U2AF65.

a Schematic diagram of RELL2 minigene and serial deletions in exon 3 and intron 3. Editing site is highlighted in red. b RT-PCR analysis of exon 3 inclusion of exogenous RELL2 transcripts in HEK293T cells that were co-transfected with the indicated minigene and overexpression construct (n = 2 biological replicates for each). c In silico prediction of RNA secondary structure by RNAfold. Base-pair probabilities are shown by a color spectrum. Asterisk indicates the editing site. d RT-PCR analysis of exon 3 inclusion of exogenous RELL2 transcripts in HEK293T cells that were co-transfected with the indicated minigene and overexpression construct (n = 2 biological replicates for each). GA-rich sequences #1 and #2 are highlighted in red and blue. e REMSA analysis of the binding of ADAR2 protein to RELL2 transcripts in vitro, using ³²P-labeled wild-type or mutant RELL2 Py-ex3 dsRNA probe with the increasing amount of recombinant ADAR2 protein. %Shift is calculated as shift band intensity over the sum of free probe and shift band intensities. f RIP-qPCR analysis of the binding of ADAR2 protein to exogenous RELL2 transcripts in vivo. WB analysis of FLAG-RIP immunoprecipitates is shown in the top panel. Input indicates 1% of the total cell lysate. Data are presented as mean ± S.D. of %input derived from qPCR technical triplicates from a representative experiment. Each dot represents a technical replicate. Statistical significance is determined by unpaired, two-tailed Student’s t-test (****P < 0.0001). g In vitro RNA–protein binding assay by UV crosslinking demonstrated the binding of U2AF65 to the RELL2 dsRNA probe, in the absence or presence of ADAR2 protein. h RNA pulldown assay detected the binding of U2AF65 protein to the dsRNA probe, in response to the addition of increasing amount of ADAR2 protein. WB analysis of U2AF65 and ADAR2 proteins in RNA pulldown (eluate) products and flow-through fractions. b, d Data are presented as the mean ± S.D. of percent spliced in (PSI) values from biological replicates. Each dot represents a biological replicate. Statistical significance is determined by paired t-test (*P < 0.05; **P < 0.01). Source data are provided as a Source Data file.