CircPCMTD1: A Protein-Coding Circular RNA That Regulates DNA Synthesis in Leukemic Myeloblasts

Introduction: The prognostic and biologic significance of circular RNAs (circRNAs) in patients with acute myeloid leukemia has recently been reported. Circular PCMTD1 (circPCMTD1) was among the circRNAs that were prognostic in this disease. Herein, we study the functional role of circPCMTD1 in leukemia.

Methods: We used RNase H-recruiting, locked nucleic acid-modified oligonucleotides (gapmers) for knock-down (KD) experiments. Cell cycle analyses were performed with bromodeoxyuridine and 7-actinomycin D staining. Apoptosis was assessed with annexin V and propidium iodide staining. Chloro-deoxyuridine (CldU) and iodo-deoxyuridine (IdU) were used for DNA fiber assays.

Results: To study the biologic function of circPCMTD1, we performed KD experiments using anti-circPCMTD1 gapmers in K562 and LAMA84 leukemia cell lines. Anti-circPCMTD1 gapmers specifically degraded the circular transcript without affecting the levels of the linear PCMTD1 (linPCMTD1). CircPCMTD1-KD led to a potent G2/M blockade and increased the percent of apoptotic blasts in both the K562 and LAMA84 cell lines. In contrast, linPCMTD1-KD did not have a significant effect on cell cycle progression or the viability of myeloblasts. Mass cytometry (CyTOF) experiments validated the cell cycle blockade after circPCMTD1-KD. In addition, CyTOF experiments identified an increase of H2AX phosphorylation, as an early downstream event of circPCMTD1-KD. We validated this finding with western blots and could also show that circPCMTD1-KD led to an increase in the phosphorylation of the ATM, ATR, CHK1, DNA-PK and RPA32 proteins. Further, we performed sequential labeling with CldU and IdU in cells treated with circPCMTD1-KD versus control, and conducted DNA fiber assays. We detected a global decrease in the length of CldU- and IdU-labeled fibers as well as of the IdU/CldU ratio in the circPCMTD1-KD cells. Taken together these data indicate that circPCMTD1-KD causes impaired DNA replication in leukemic cells.

To identify proteins with which circPCMTD1 interacts, we performed ribonucleoprotein complex purification and comparative proteomics analyses, using the RAP-MS technique (McHugh et al, Nature 2015;521:232). We identified METTL3 as a strong candidate binder of circPCMTD1. We validated the METTL3-circPCMTD1 interaction by RNA Immunoprecipitation experiments. We also found enrichment of circPCMTD1 in capture experiments from total RNA with an anti-N⁶-methyladenosine (m⁶A) antibody compared to control. METTL3-mediated, high m⁶A status has been associated with protein-coding potential of circRNAs (Yang et al, Cell Res 2017;27:626). To evaluate whether circPCMTD1 interacts with the translational machinery of the leukemic blasts, we performed sucrose gradient fractionation and targeted polysome profiling and we found enrichment of the circPCMTD1 in the 80S monosomes, and both light and heavy molecular weight polysomal fractions. In search of an open reading frame, we identified a stop codon in the 5' UTR of exon 2 of the PCMTD1 transcript. While this stop codon should have no functional relevance for the linPCMTD1, it was in-frame with a start codon also contained in the circPCMTD1. We generated an antibody against a region specific for the circPCMTD1-derived peptide. The antibody detected a protein of 35 kD, which was depleted after circPCMTD1-KD. We next performed immunoprecipitation experiments followed by mass spectrometry analysis, using our custom antibody. These showed a strong and specific enrichment of the BLM, TOP3A and RMI1 proteins in the circPCMTD1 samples. Each interaction was validated with reciprocal co-immunoprecipitation experiments. BLM, TOP3A and RMI1 form the BTR complex, which is implicated in DNA replication and repair. CircPCMTD1-KD had no effect on the total amount of each of the three proteins but reduced their interaction and the amount of the formed BTR complex. Biotin labeling and capture of newly synthetized DNA with the iPOND technique showed a significant decrease in the presence of the BTR complex on the sites of active DNA replication after circPCMTD1-KD.

Conclusions:CircPCMTD1 is a circRNA with protein-coding potential. The circPCMTD1-derived peptide interacts with BLM, TOP3A and RMI1 and facilitates the formation of the BTR complex. In this context, circPCMTD1 is indispensable for the proliferation of leukemic myeloblasts as circPCMTD1-KD impairs DNA replication.