Fig. 4: LNC-SNO49AB binds ADAR1 in the nucleolus.
From: The snoRNA-like lncRNA LNC-SNO49AB drives leukemia by activating the RNA-editing enzyme ADAR1
a Representative images of dcas13-mediated LNC-SNO49AB labeling (green); LNC-SNO49AB colocalized with FBL (red) in NB4 cells. Scale bars, 2 μm. b Subcellular localization of LNC-SNO49AB in MV4-11 and NB4 cells. MALAT1, SNORD49A and GAPDH RNAs were used as controls for nucleolar, nuclear, and cytoplasmic fractions, respectively. c Silver staining of proteins pulled down by in vitro transcribed tRSA and tRSA-LNC-SNO49AB from total protein extracts of MOLM13 cells. Red arrow shows the specific band. d ADAR1 was pulled down by tRSA-LNC-SNO49AB but not tRSA in MOLM13, MV4-11 and RS4;11 cells, as determined by western blotting. e In vitro RNA pull-down assay with purified recombinant Flag-ADAR1 and tRSA-LNC-SNO49AB. Representative results are shown from three replicates. f Representative images of dcas13-mediated LNC-SNO49AB labeling (green); LNC-SNO49AB colocalized with ADAR1 (red) in NB4 cells. Scale bars, 2 μm. g Schematic representation of the ADAR1 deletion mutants used in the RNA pull-down and RIP assays shown in h and i. h Western blot of the indicated Flag-tagged ADAR1 deletion mutants retrieved by in vitro transcribed tRSA-LNC-SNO49AB obtained from 293T cell extracts. *Represented the non-specific band. Representative results are shown from three replicates. i qRT-PCR analysis of endogenous LNC-SNO49AB enriched by the indicated Flag-tagged ADAR1 deletion mutants in 293T cells. GAPDH mRNA served as the negative control. Representative results are shown from three replicates. j Schematic representation of the ADAR1 deletion mutants used in the RNA pull-down and RIP assays shown in k–m. k qRT-PCR analysis of endogenous LNC-SNO49AB enriched by the indicated Flag-tagged ADAR1 deletion mutants in 293T cells. GAPDH served as the negative control. Representative results are shown from three replicates. l qRT-PCR analysis of endogenous LNC-SNO49AB enriched by the indicated Flag-tagged ADAR1 deletion mutants in 293T cells. PTPN6, CALM1 and APH1B are the representative targets of ADAR1. Representative results are shown from three replicates. m Western blot of the indicated Flag-tagged ADAR1 deletion mutants retrieved by in vitro transcribed tRSA-LNC-SNO49AB obtained from 293T cell extracts. Representative results are shown from three replicates.
