Distinct RNA structural domains cooperate to maintain a specific cleavage site in the 3 '-UTR of IGF-II mRNAs

The insulin-like growth factor II mRNAs are targets for site-specific endon ucleolytic cleavage in the 3'-UTR, which results in a very stable 3' cleava ge product of 1.8 kb, consisting of 3'-UTR sequences and a poly(A) tail. Th e 5' cleavage product contains the coding region and is rapidly degraded. T hus, cleavage is thought to provide an additional way to control IGF-EI pro tein synthesis. We had established that cleavage requires two widely separa ted sequence elements (I and II) in the 3'-UTR that form a stable duplex of 83 nucleotides. The cleavage-site itself is located in an internal loop pr eceded by two stable stem-loop structures. Furthermore, in a study which wa s based on RNA folding algorithms, we have shown that there are specific se quence and structural requirements for the cleavage reaction. Here, the fun ctions of the different structural domains in cleavage were assessed by del etion/mutational analyses, and biochemical structure probing assays were pe rformed to characterize better the RNA structures formed and to verify the computer folding predictions. The data suggest that the stem-loop domain co ntributes to maintain a highly specific c leavage-site by preventing the fo rmation of alternative structures in the cleavage-site domain. Involvement of the nucleotides in the cleavage-site loop itself in non-Watson-Crick int eractions may be important for providing a specific recognition surface for an endoribonuclease activity. (C) 2000 Academic Press.