Characterization of a protein interaction with the proopiomelanocortin mRNA: An RNA mutagenesis analysis

Proper regulation of the proopiomelanocortin (POMC) gene is critical to the mammalian stress response. The POMC gene is translationally controlled, and this regulation may contribute to the rapidity by which an organism can respond to physical or psychological stressors. Previous work in our laboratory has led to the proposal of a mechanism by which POMC may be translationally controlled. In this model, cytoplasmic pituitary protein binds to a stem-loop structure within the POMC mRNA coding region, to form multiple RNA-protein complexes, the most prominent of which has an apparent molecular weight of 55 kDa. The formation of this RNA-protein complex is thought to inhibit POMC translation. To test this mechanistic model and to further define the RNA-protein interactions that may be important to POMC gene regulation, mutations were created in the POMC mRNA. RNAs that were mutant in the single-stranded bulge or loop regions of the stem-loop or that were unable to form a stem bound rat pituitary protein in RNA gel mobility shift and UV-crosslinking assays in a manner similar to the wild-type stem-loop. To further examine the requirements of complex formation, large deletions ranging in size from 100 to 800 nucleotides in length were made throughout the POMC mRNA. Each deletion mutant tested, including a POMC RNA which lacks the stem-loop, retained the ability to form the 55 kDa complex in UV-crosslinking assays. These results demonstrate that the POMC stem-loop structure is sufficient, but not necessary for formation of the 55 kDa complex. The RNA requirements for 55 kDa complex formation are less specific than previously proposed and may be fulfilled by RNA sequences that possess an indeterminate combination of length and structure. Additional studies were performed to test the effect of POMC mutations on extract-dependent translational inhibition. These in vitro translation experiments could not be completed because we were unable to replicate conditions where wild-type POMC was specifically inhibited by extract. The findings from this work have led to a modification of our mechanistic model for POMC translational regulation