Exploring structural determinants and the role of nucleolin in formation of the long-range interactions between untranslated regions of p53 mRNA

p53 protein is a key regulator of cellular homeostasis by coordinating the framework of antiproliferative pathways as a response to various stress factors. Although the main mechanism of stress-dependent induction of p53 protein relies on post-translational modifications influencing its stability and activity, a growing amount of evidence suggests that complex regulation of p53 expression occurs also at the mRNA level. This study explores structural determinants of long-range RNA-RNA interactions in p53 mRNA, crucial for stress-dependent regulation of p53 protein translation. We demonstrate that the 8-nt bulge motif plays a key structural role in base-pairing of complementary sequences from the 5' and 3' untranslated regions of p53 mRNA. We also show that one of the p53 translation regulators, nucleolin, displays an RNA chaperone activity and facilitates the association of sequences involved in the formation of long-range interactions in p53 mRNA. Nucleolin promotes base-pairing of complementary sequences through the bulge motif, because mutations of this region reduce or inhibit pairing while compensatory mutations restore this interaction. Mutational analysis of nucleolin reveals that all four RNA recognition motifs are indispensable for optimal RNA chaperone activity of nucleolin. These observations help to decipher the unique mechanism of p53 protein translation regulation pointing to bulge motif and nucleolin as the critical factors during intramolecular RNA-RNA recognition in p53 mRNA.