Figure 1.

The HNRPLL pre-mRNA transcript is differentially edited in tumors and a substrate of ADAR1 and ADAR2. A and B, editing profiles of HNRPLL in clinical tumor specimens. Distribution of the A-to-G editing rates of three editing sites identified in the HNRPLL transcript is shown for paired kidney (a; n = 34) and bladder (b; n = 16) tumor patient samples. C, an exon E12A-containing HNRPLL variant was detected by end point PCR, using primers as shown by the schematic representation in the upper panel. PCR amplicons from the indicated cell lines were sequenced by the Sanger method shown in the lower panel, and the marked nucleotides denote the A-to-I(G) editing positions. D and E, vectors expressing control (pSUPER) or ADAR1/2-specific (shADAR1/2) shRNAs were transfected into HeLa cells, and protein knockdown in transfected cells was assessed by Western blotting, with GAPDH and β-actin as loading control. E12A editing in the transfected cells was determined by Sanger sequencing. F, for RNA-IP assay, HeLa cell lysates were immunoprecipitated with control IgG or ADAR1 antibody, and the precipitated RNA was measured by qPCR assay with specific primers to GAPDH and pre-mRNA of HNRPLL (n = 3). G, HeLa cells were transfected with the indicated plasmids and harvested for immunoprecipitation with anti-FLAG M2 beads. The RNA abundance in precipitated complexes was determined by a qPCR assay with specific primers to HNRPLL pre-mRNA, reference HNRPLL mRNA (WT), and the E12A variant (n = 3). H, Sanger sequencing of the genomic DNA region corresponding to the editing positions. The highlighted nucleotides indicate positions undergoing editing in the transcripts.