STABILITY DETERMINANTS ARE LOCALIZED TO THE 3'-UNTRANSLATED REGION AND 3'-CODING REGION OF THE NEUROFILAMENT LIGHT SUBUNIT MESSENGER-RNA USING A TETRACYCLINE-INDUCIBLE PROMOTER

The tetracycline-responsive expression system of Bujard was used to co mpare rates of decay of wild-type and mutant neurofilament (NF) Light subunit (NF-L) mRNAs, Optimal conditions for activation and inactivati on of the target transgene were determined using a luciferase reporter gene. Analyses of mRNA stability were thereupon conducted on cells th at were doubly transfected with transactivator and inducible target ge nes and derived from pooled clones of transfected cells. Rates of mRNA decay were compared upon inactivation of the transgenes after high le vels of mRNA had been induced. Deletion of the 445-nucleotide (nt) 3'- untranslated region (3'-UTR) (L/+++-) or 527 nt of the 3'-coding regio n (3'-CR) (L/++-+) increased the stability of NF-L mRNA compared with the full-length (L/++++) transcript in neuronal (N2a and P19 cells) an d non-neuronal (L cells) lines. Deletion of both the 3'-UTR and 3'-CR (L/++--) led to a further stabilization of the transcript. A major sta bility determinant was then localized to a 68-nt sequence that forms t he junction between the 3'-CR and 3'-UTR of NF-L and is the binding si te of a unique ribonucleoprotein complex (Canete-Soler, R., Schwartz, M. L., Hua, Y., and Schlaepfer, W. W. (1998) J. Biol. Chem. 273, 12655 -12661). The studies establish a novel system for mapping determinants of mRNA stability and have applied the system to localize determinant s that regulate the stability of the NF-L mRNA.