Multiple splicing variants of Naf1/ABIN-1 transcripts and their alterations in hematopoietic tumors

Nafl (Nef-associated factor 1)/TNIP1/ABIN-1 (A20-binding inhibitor of NF-κB activation) is a cellular protein that interacts and cooperates with the NFκB inhibiting protein A20. It is reported that Nafl attenuates epidermal growth factor (EGF)/extracellular- signal-regulated kinase2 (ERK2) nuclear signaling. Nafl also binds to Nef, which plays a key role in acquired immunodeficiency syndrome pathogenesis and HIV-1 virus replication. Nafl mRNA consists of 18 exons and multiple splice variants have been reported; two isoforms for exon 1, deletion of exon 2 and isoforms a and B for exon 18. Using specimens from 29 acute myeloid leukemia (AML) patients, we detected a high frequency of allelic loss on DNA at STS marker D5S2014 near the Nafl gene. We therefore performed mutation and expression analyses using leukemia-lymphoma lines and 6 pairs of clinical AML samples. There was no mutation in the Nafl coding region of any sample. As a result of expression analysis, we identified novel splice variants of the Nafl gene; deletion of exon 16 (Nafl a2, Nafl B2), deletion of exon 16 with an insertion (Nafl α3, Nafl B3) and deletion of exons 16 and 17 (Nafl a4). Nafl a3 and β3 showed premature termination. In peripheral blood mononucleocytes (PBMNCs) from healthy adults, almost no expression of full-length Nafl (Nafl FL ), Nafl a3 and B3 were observed. In contrast, their expression was clear in AML blasts and in the majority of leukemia-lymphoma lines investigated. Nafl a2 was widely expressed in PBMNCs from healthy adults, AML blasts and cell lines, suggesting it is the main transcript of the Nafl gene. Luciferase assay revealed that Nafl a2 had equal NF-KB inhibitory effect to that of Nafl FL , while Nafl a4 was less effective. In clinical AML patients, the expression of Nafl a3 was much higher at diagnosis than on remission after chemotherapy, suggesting the possible dominant negative effect of Nafl a3.