Abstract
The human major histocompatibility complex (MHC) class I related chain B (MICB) gene encodes a membrane-bound glycosylated protein which is a ligand for the natural killer (NK) cell receptor NKG2D. Binding of MICB to NKG2D activates the cytolytic response of NK cells. MICB, like its close functional homolog MICA, is expressed selectively by cells undergoing malignant transformation or exposed to stress such as heat shock or viral infection. Nevertheless, unlike MICA, MICB mRNA transcripts are unstable, which may contribute to the low level of MICB protein expression in cells. In the present study, I has determined that endogenous MICB mRNA degrades 8-times faster than MICA mRNA. Next, since MICB mRNA carries a much longer 3’ untranslated region (UTR) (1229bp) than MICA mRNA (175bp), I hypothesized that the stability of MICB mRNA was affected by its unusually long 3’UTR. Real-time RT-PCR analysis of COS-7 cells transfected with a series of MICB 3’UTR deletion mutants showed an inverse correlation between the length of MICB 3’UTR and MICB mRNA stability where the proximal end of MICB 3’UTR appeared to play a critical role. These findings were corroborated by the results of flow cytometric and Western blot analyses of MICB protein expression in COS-7 and HEK293FT cells transfected with MICB 3’UTR deletion mutants. As a result, a potential regulatory region between bp194 and bp361 of the proximal MICB 3’ UTR was identified. Lastly, the conservation of the MICB mRNA 3’UTR regulatory function was indicated by the markedly reduced expression of enhanced green fluorescence protein (eGFP) encoded by the eGFP construct engineered with the 1152 bp MICB 3’ UTR. Elucidation of the role of MICB 3’ UTR in the regulation of MICB expression will improve our understanding of how the status of MICB protein is altered in cells by a post-transcriptional mechanism. This information may suggest ways to increase MICB mRNA stability and protein production in cells, making them a better cytolytic target for NK cells.