Post-transcriptional regulation of human pulmonary surfactant protein B (SP-B) mRNA
Evidence indicates that stability of human pulmonary surfactant protein B (hSPB) mRNA is regulated by glucocorticoids via mRNA:protein interactions at the 3'-untranslated region (3'-UTR). To map stability determinants to specific elements, 30-nucleotide replacement mutants were generated using scanning mutagenesis, and cloned into a novel bicistronic GFP plasmid under the control of a CMV promoter. Steady-state levels of hSP-B mRNA in the absence or presence of dexamethasone (Dex) were determined by Northern blot analysis. Several instability elements were identified in the 5'-most region (hSP-B7.6 region) of the hSP-B mRNA 3'-UTR, and these elements were necessary and sufficient to impart regulation on intrinsic and Dex-modulated mRNA stability, regardless of cell type. More importantly, it may be the stem-loop structure and the A-rich stretch that grant the 002 element its highly destabilizing nature and its ability to mediate Dex stabilization. Despite the stabilizing effect of Dex, glucocorticoid receptor does not seem to participate in Dex-augmented half-life of hSP-B mRNA. Dex may regulate the stability of hSP-B mRNA via a nongenomic mechanism. This report is the first to describe the usefulness of a novel bicistronic plasmid for in vivo stability and functionality assays, and to demonstrate the feasibility of steady-state mRNA levels to represent mRNA stability. The possible interference by transcription inhibitors commonly used in half-life experiments is therefore eliminated.
Huang, Helen Wan-ching, "Post-transcriptional regulation of human pulmonary surfactant protein B (SP-B) mRNA" (2007). Texas Medical Center Dissertations (via ProQuest). AAI3287848.