Chimeric antisense RNA derived from chromosomal translocation modulates target gene expression

Chromosomal translocations in hematologic malignancies1 are closely related to the molecular pathogenesis. Usually, the directions of the two genes involved in the chromosomal translocations are the same, resulting in chimeric proteins that retain their functional domains. Here we report a chromosomal translocation from a myelodysplastic syndrome (MDS) patient resulting in a fusion gene consisting of the sense strand of the TEL/ETV6 gene on 12p13 fused with the antisense strand of the Thousand-and-one amino acid protein kinase 1 (TAOK1) gene on 17q11. We suggest the possibility that the chimeric transcript may act as an antisense RNA on wild-type TAOK1 mRNA, resulting in downregulation of TAOK1 protein expression.

A 73-year old man was admitted to our hospital because of severe anemia (hemoglobin 5.5 g/dL) and thrombocytopenia (platelets 72×10/L). Bone marrow aspiration indicated dysplasia in three lineages with 11% blasts. The patient was diagnosed with MDS-RAEB-II. Chromosomal analysis of bone marrow cells revealed t(12;17)(p13;q11). Four months after diagnosis, disease progression to acute myelogenous leukemia was confirmed without additional chromosomal abnormalities. Conventional chemotherapy was performed, but he died of leukemia progression nine months after diagnosis.

Using leukemia cells at diagnosis, fluorescent in situ hybridization (FISH) analysis showed a split signal of TEL/ETV6 gene (Figure 1A and B). The TEL/ETV6 fusion transcript was amplified by 3′-RACE2 and analyzed with DNA sequencing (Online Supplementary Appendix). The 3′ end of the exon 2 of TEL/ETV6 was fused with antisense sequences of intron 19 of TAOK1, which was followed by antisense sequences of exon 19 and intron 18 of TAOK1 (Figure 1C) (GenBank #JN603181). Amino acid sequences of the carboxyl (C)-terminus of exon 2 of TEL/ETV6 were followed by nonsense sequences derived from the antisense TAOK1 sequences of intron 19 (Figure 1C and D).

Expression of the fusion transcript (TEL-TAOK1ap) was checked in primary bone marrow cells from MDS patients and cell lines by RT-PCR (TT-U and TT-L primers in Figure 1D), and showed that only the patient’s sample that held t(12;17) expressed the TEL-TAOK1ap transcript (Figure 2A, lane 1). Expression of the wild-type (WT) TAOK1 protein was confirmed with immunoblotting using whole-cell lysates from cell lines and primary bone marrow cells from MDS patients (Figure 2B), indicating that the level of WT-TAOK1 protein expression was much lower in the patient’s cells that held t(12;17) (lane 8) than in normal bone marrow cells (lane 1) and several leukemia cell lines (lanes 2–7). Samples from other MDS patients (lanes 9–13) were analyzed; some patients also showed lower expression of WT-TAOK1 protein (lanes 9 and 10). Expression levels of TAOK1 mRNAs were confirmed with quantitative (real-time) RT-PCR using the 3′ and 5′ region-specific probe sets for WT-TAOK1 transcripts (Figure 2C). Expression of 5′- and 3′-TAOK1 was lower in the t(12;17) patient’s cells than in leukemia cell lines (Figure 2D). The expression levels of 5′ and the 3′ TEL/ETV6 mRNAs were mostly similar in the patient’s cells in semi-quantitative RT-PCR analysis (data not shown). These data suggest that WT-TAOK1 protein expression is down-regulated in some MDS cases by unknown molecular mechanisms.

Figure 1.Aberrant fusion transcript derived from chromosomal translocation t(12;17)(p13;q11). (A) FISH probes for TEL/ETV6. (B) A split signal was observed in the patient’s leukemia cells. Red signal is from the TEL-5′ probe, and green signal is from the TEL-3′ probe. Non-split signal from wild-type TEL/ETV6 is observed as a yellow dot. (C) DNA sequence of the chimeric transcript (TEL-TAOK1ap) is shown in black letters. Amino acid sequence from exon 2 of TEL/ETV6 is indicated in red letters. Amino acid sequence from the antiparallel sequence of intron 19 of TAOK1 is indicated in blue letters. Note that amino acid sequence in blue letters is completely different from the sequence of WT-TAOK1 protein. (D) Schematic representation of genomic structure around the breakpoint. Tel: telomere; cent: centromere; ap: antiparallel sequence; Ex; exon, *: stop codon; pA: poly adenine tail, black boxes; exons, shaded boxes; untranslated exon, white boxes; intron sequences that are utilized as exons in TEL-TAOK1ap.

Next, we hypothesized that the antiparallel portion of exon 19 of TAOK1 in TEL-TAOK1ap transcript may act as an antisense RNA to knock-down WT-TAOK1 mRNA expression. To test this hypothesis, the TEL-TAOK1ap transcript was over-expressed in 293T cells. As a control, shRNA for TAOK1 mRNA was transfected into 293T cells, resulting in a significant reduction in endogenous TAOK1 protein (Figure 2E). When using the TEL-TAOK1ap expression vector in 293T cells, endogenous TAOK1 protein expression was reduced in a dose-dependent manner (Figure 2F). These findings support our hypothesis that TEL-TAOK1ap has an RNA-interfering effect on WT-TAOK1 mRNA.

Figure 2.Expression of TEL-TAOK1ap chimeric transcript and its RNA interfering effect in regulating endogenous TAOK1 expression. (A) TEL-TAOK1ap transcript and (B) WT-TAOK1 protein expression in the patient’s primary cells and human leukemia cell lines. RT-PCR (RT) (A) and immunoblotting (IB) using an anti-TAOK1-C-terminus antibody (B) were performed. The patient's samples holding t(12;17)(p13;q11) are indicated as t(12;17). Primary patient’s bone marrow cells were used in lanes 1 to 9 in (A) and lanes 8 to 13 in (B). Beta-actin and GAPDH are the positive controls for RT-PCR and immunoblotting, respectively. (C) Schematic representation of WT-TAOK1 mRNA. Shaded boxes indicate probes for quantitative RT-PCR. Black triangles indicate break point in the TEL-TAOK1ap fusion. (D) Quantitative RT-PCR using TAOK1-specific probes as indicated in (C). (E) Expression vector for shRNA against TAOK1 mRNA or control mock vector (3 mg each) was transfected into 293T cells (5×10⁵ cells). Immunoblotting for endogenous TAOK1 was performed using anti-TAOK1 C-terminus antibody. (F) Expression vector for TEL-TAOK1ap (0, 0.5, 1, and 3 μg in each sample) or the control mock vector (3, 2.5, 2, and 0 μg in each sample) was transfected into 293T cells (5×10⁵ cells). Endogenous TAOK1 protein expression (upper panel) and over-expressed TEL-TAOK1ap transcript (third panel) were confirmed by IB and RT. Note that the endogenous TAO1 protein expression level was decreased in a dose-dependent manner. MDS/AML; acute myelogenous leukemia followed by MDS; RAEB: refractory anemia with excess blasts; RCMD: refractory cytopenia with multilineage dysplasia; RCUD: refractory cytopenia with unilineage dysplasia; CMMoL: chronic myelomonocytic leukemia; UTR: untranslated region; CC: coiled-coil domain.

A previous report showed that translocation of t(12;17)(p13;p12-p13) in secondary AML results in fusion of TEL/ETV6 and the antisense strand of PER1.3 Expression of the chimeric transcript containing anti-sense sequences to PER1 was confirmed in this case. Recently, RNA interfering activity by small non-coding RNAs, such as small interfering RNA, micro-RNA, and PIWI-RNA,4 has been reported. Furthermore, several reports have indicated that long non-coding RNAs5 and natural antisense transcripts6 play crucial roles in regulating mRNA expression of target genes. Our findings suggest a mechanism in which a chimeric transcript regulates target gene expression via an RNA interfering effect.

The TEL-TAOK1ap chimeric transcript may have dual functions, including an antisense effect to interfere with WT-TAOK1 mRNA and production of C-terminally-truncated TEL/ETV6. A previous report indicated that TAOK1 is a serine/threonine kinase that plays an important role in the p38 MAPK signaling pathway.7 Knockdown of TAOK1 in HeLa cells disrupts normal cell division due to a disorder in the spindle checkpoint function.8 In our experiment (Figure 2B), some MDS patients showed relatively lower expression of WT-TAOK1 protein compared with acute leukemia cell lines, suggesting that lower expression might be related to the pathogenesis of MDS, such as aberrant cell division and/or dysplasia. Furthermore, there are many reports of TEL/ETV6 fusion proteins that contain the N-terminus of TEL/ETV6 in leukemia patients,9 suggesting that aberrant substitution or truncation of the C-terminus of TEL/ETV6 may contribute to leukemia biology. The biological significances of TEL-TAOK1ap chimeric transcripts and their relationship with MDS/leukemia genesis require further study.

Footnotes

• Funding: this work was supported by Grants-in-Aid from the Ministry of Scientific Research of Education, Culture, Sports, Science and Technology, and the Ministry of Health, Labor and Welfare, and the Japanese Society for the Promotion of Science (117100000439). We are indebted to Chika Wakamatsu, Eriko Ushida, Mari Otsuka, and Yukie Konishi for their valuable assistance in the laboratory.
• The online version of this article has a Supplementary Appendix.
• The information provided by the authors about contributions from persons listed as authors and in acknowledgments is available with the full text of this paper at www.haematologica.org.
• Financial and other disclosures provided by the authors using the ICMJE (www.icmje.org) Uniform Format for Disclosure of Competing Interests are also available at www.haematologica.org.