Post-transcriptional regulation of ULBP1 ligand for the activating immunoreceptor NKG2D involves 3' untranslated region

The stress-inducible ULBP1 cell surface ligand for the activating immunoreceptor NKG2D allows recognition and lysis of tumor cells by natural killer (NK) and T cells. Understanding of mechanisms regulating ULBP1 expression is limited, but it is important for exploiting NKG2D-dependent antitumor responses. We studied the role of 3' untranslated region (3' UTR) in post-transcriptional regulation of ULBP1 expression in Jurkat and HeLa cells. Analysis of 2.4 kb-long 3' UTR revealed the presence of four AU-rich elements (ARE) and more then 200 putative microRNA binding sites. Stable or transient delivery of luciferase reporter constructs containing ULBP1-3' UTR sequences resulted in a strong reduction of luciferase activity to 7-22% with the full-length 3' UTR or 19%-62% with its fragments, indicating a contribution of 3' UTR to regulation of ULBP1 gene. Mutations introduced to ARE motifs significantly diminished luciferase activity, suggesting mRNA stabilizing effect of ARE. Among ULBP1-specific candidate microRNAs, we found miR-140-5p/-409-3p/-433-3p/-650 expressed in HeLa and Jurkat cells, and the microRNA involvement was supported by luciferase reporter assays with constructs carrying seed sequence mutations. However, microRNA overexpression or partial silencing of the microRNA processing enzyme Drosha did not equivocally clarify the role of microRNAs in regulation of ULBP1. Altogether these results provide evidence for a novel 3' UTR-mediated mechanism of regulation of ULBP1 at the post-transcriptional level.