Extended Data Figure 2: Identification of the ALKATI transcript.
From: Alternative transcription initiation leads to expression of a novel ALK isoform in cancer
a, Northern blot of wild-type ALK-expressing neuroblastoma cell lines (SK-N-DZ and SK-N-BE2), EML4–ALK-expressing lung cancer cell lines (H3122, variant (V) 1 and H2228, variant (V) 3), ALKATI-expressing melanoma, one anaplastic thyroid carcinoma (ATC-28), and negative controls (melanoma cell lines). Except for the negative controls, each lane shows two bands: the lower B-band matches the shorter canonical (RefSeq) ALK transcript ending at ∼chr2:29,415,640; the upper A-band corresponds to a transcript with a 1.8 kb longer 3′-UTR ending at ∼chr2:29,413,840. Two ALKATI-expressing melanomas, MM-284 and MM-74, show only weak signals because less than 1 μg RNA was available; for all other samples 5–10 μg RNA were used. See Supplementary Fig. 1 for uncropped blots. b, RNA-seq data displayed in IGV. The Sashimi plot illustrates the shorter B and the longer A ALK transcripts by the sharp drop of sequencing reads in the 3′-UTR at chr2:29,415,640 for the B and at chr2:29,413,840 for the A transcript. c, IGV view of the 5′-RACE-cDNA fragments obtained by massively parallel sequencing. More than 95% of the sequencing reads (grey arrows) start within the main ATI site of 25 bp (hg19 chr2:29,446,744–29,446,768). d, Sanger sequencing of the cloned 5′-RACE-cDNA fragments confirms the continuous transcription starting in ALK intron 19 and extending to exons 20 and 21. e, The ALKATI transcript consists of ∼400 bp upstream of exon 20 and of ALK exons 20–29. The transcriptional initiation site was defined as the first base pair at which more than 5% of the transcripts were initiated (chr2:29,446,766). Other major transcription initiation sites are marked in red, the 5′- and 3′-UTRs in dark blue, the coding DNA sequence (CDS) in black, and the first and last base of each exon in light blue. The translation is initiated at 3 start codons (ATGs; bold and underlined): first ATG, hg19 chr2:29,446,360–29,446,362; second ATG, (+ 7–9); and third ATG (+ 61–3). f, The amino acid sequence of ALKATI. The translation is initiated at 1 of 3 start codons. The corresponding 3 methionines (bold and underlined) result in 3 different proteins, 61.08 kDa (552 amino acids), 60.82 kDa (550 amino acids), and 58.71 kDa (532 amino acids). The kinase domain is highlighted in red. The lysine in the ATP binding domain is marked bold and underlined, and was mutated to methionine (referring to wild-type ALK: p.K1150M) in the kinase-dead ALKATI-KD.
