Figure 1. RT–PCR identification of an abnormal transcript of the PTPN6 gene in normal and leukemic bone marrow cells and cell line. (a) Diagrammatic representation of the human PTPN6 locus organization as previously described (45). P1, promoter active in non-hematopoietic cells; P2, hematopoietic cell-specific promoter. PTPN6 cDNA was PCR amplified using the primers shown at the top of the diagram of the predicted protein structure. A schematic representation of the 5′ end of the PTPN6 abnormal product detected by RT–PCR using oligonucleotides PTP-141 and PTP-897 is shown below the genomic organization. Retention of the 251 bp intron 3 is arrowed and the 3′ splice junction neighbor sequence given below. The asterisked adenosine residue is the presumptive site of the branch formation subjected to editing. The sequence chromatogram of cDNA clone E4.18 obtained from the bone marrow blasts of a patient with acute myeloid leukemia (AML-34) shows the editing A→G conversion, which is absent in genomic DNA. (b) Agarose gel analysis of the PCR products obtained on cDNAs prepared from the bone marrow (BM) and peripheral blood (PB) blasts of patient AML-34 at diagnosis, from the bone marrow of healthy donors and from HL-60 cells. Using the PTP-141 and PTP-897 primers that amplify a normal 757 bp cDNA fragment, an additional 1008 bp fragment with the 251 bp intron 3 can be seen between nucleotides 275 and 276. The DNA marker is a 100 bp DNA ladder (M). From: RNA hyperediting and alternative splicing of hematopoietic cell phosphatase (PTPN6) gene in acute myeloid leukemia Hum Mol Genet. 2000;9(15):2297-2304. doi:10.1093/oxfordjournals.hmg.a018921 Hum Mol Genet |

Figure 2. Nucleotide sequence of the 5′ end of the 1008 bp PTPN6 abnormal product detected by means of RT–PCR using oligonucleotides PTP-141 and PTP-897. The sequence of the retained intron 3 is shaded. A total of seven A→G edited sites (indicated by the underlined adenine residues) were detected analyzing 25 independent clones: only one of these sites was located within intron 3. This RNA editing destroys the branch formation site, making it unrecognizable to the splicing machinery. The translation initiation codon is indicated by double underlining and the exon junctions by triangles (Δ). A potential 44 amino acid protein is terminated by a UAG stop codon (asterisked) arising from the retention of intron 3 in the edited cDNA clones. A pair of in-frame potential translation reinitiation codons downstream of the premature stop codon are indicated in bold. From: RNA hyperediting and alternative splicing of hematopoietic cell phosphatase (PTPN6) gene in acute myeloid leukemia Hum Mol Genet. 2000;9(15):2297-2304. doi:10.1093/oxfordjournals.hmg.a018921 Hum Mol Genet |

Figure 3. Semi-quantitative RT–PCR on CD34⁺/CD117⁺ bone marrow blasts at diagnosis and remission. (a) Semi-quantitative RT–PCR on bone marrow from healthy donors (BM-MI4238 and BM-MI1032), HL-60 cells and CD34⁺/CD117⁺ BM blasts from seven acute myeloid leukemia patients at diagnosis and remission. The normal 206 bp and the alternatively spliced 457 bp RT–PCR products and correspondent peak profiles representing the quantitative measurements are shown for each sample. Comparison of results obtained on bone marrow blasts at diagnosis (D) and follow-up (F) is shown for patients AML-17, AML-23, AML-32, AML-7 and AML-21. RT–PCR results on bone marrow at diagnosis from patient AML-2 and a comparison of bone marrow (BM) and peripheral blood (PB) at diagnosis from patient AML-34 are shown. (b) Range of aberrant spliced PTPN6 transcript levels in normal bone marrow compared with BMMNCs at diagnosis and remission state. Quantitative analysis of alternatively spliced transcripts was obtained by capillary electrophoresis of RT–PCR products (a) and quantitative measurements using an ABI 310 genetic analyzer and Gene Scan software. For each sample the quantitative measurement of the alternatively spliced 457 bp product was divided by the quantitative measurement of the normal 206 bp plus the alternatively spliced 457 bp products yielding the percentage of the intron-retained transcript which is plotted on the graph. Alternative spliced transcript levels are higher in leukemic blasts at diagnosis in comparison with BMMNCs at remission and are lower in normal donors and HL-60 cells. From: RNA hyperediting and alternative splicing of hematopoietic cell phosphatase (PTPN6) gene in acute myeloid leukemia Hum Mol Genet. 2000;9(15):2297-2304. doi:10.1093/oxfordjournals.hmg.a018921 Hum Mol Genet |

Figure 4. Poisoned primer extension assay of the aberrant PTPN6 splicing fragment from AML blasts. (a) Schematic representation of the primer extension assay. The end-labeled extension primer is extended by Vent(exo-) DNA polymerase (New England Biolabs, Beverly, MA) to a 25mer when the cDNA is generated from an unedited template, but to a 34mer if the template is edited. (b) Poisoned primer extension assays were carried out on gel-excised aberrant spliced cDNA fragments. The autoradiograph shows the unextended primer, the first stop produced by unedited PTPN6 cDNA, and the second stop produced by edited PTPN6 cDNA for all of the AML samples analyzed. Clones D2.5 and E4.18 [whose origin is detailed in (c)] are included as negative (C–) and positive (C+) controls. (c) Two representative poisoned primer extension assays of cloned PTPN6 cDNA: E4.18 is a clone containing the edited G⁷⁸⁶⁶, whereas D2.5 is a clone which reverted the A→G modification to normal by means of site-directed mutagenesis on E4.18. From: RNA hyperediting and alternative splicing of hematopoietic cell phosphatase (PTPN6) gene in acute myeloid leukemia Hum Mol Genet. 2000;9(15):2297-2304. doi:10.1093/oxfordjournals.hmg.a018921 Hum Mol Genet |

Figure 5. In vitro splicing of SP6/PTPN6 RNA from clones E4. 18 and D2 Figure 5.In vitro splicing of SP6/PTPN6 RNA from clones E4.18 and D2.5 containing intron 3. (a) The sequence of the flanking nucleotides of the putative branch site of the PTPN6 transcript within intron 3 is compared with that of the human β-globin intron 1 branch site and the branch site consensus sequence motif. The exon and intron sequences are respectively indicated by upper and lower case lettering. The correspondence of the compared sequence at the branch formation site is boxed. (b) Association of PTPN6 IVS3 splicing with RNA editing. The aberrant PTPN6 splice RNAs isolated from the E4.18 and D2.5 clones were subjected to in vitro splicing reactions in the presence (+) or absence (–) of HelaSplice nuclear extract. RT–PCR with PTP-141 and PTP-346 primers revealed two PCR products in the case of the D2.5 clone (a 457 bp fragment containing intron 3 and a 206 bp fragment representing the normal splice product), but no splicing activity in the case of the E4.18 clone. From: RNA hyperediting and alternative splicing of hematopoietic cell phosphatase (PTPN6) gene in acute myeloid leukemia Hum Mol Genet. 2000;9(15):2297-2304. doi:10.1093/oxfordjournals.hmg.a018921 Hum Mol Genet |