Stability determinants are localized to the 3'-untranslated region and 3'-coding region of the neurofilament light subunit mRNA using a tetracycline-inducible promoter.
The tetracycline-responsive expression system of Bujard was used to compare rates of decay of wild-type and mutant neurofilament (NF) light subunit (NF-L) mRNAs. Optimal conditions for activation and inactivation of the target transgene were determined using a luciferase reporter gene. Analyses of mRNA stability were thereupon conducted on cells that were doubly transfected with transactivator and inducible target genes and derived from pooled clones of transfected cells. Rates of mRNA decay were compared upon inactivation of the transgenes after high levels of mRNA had been induced. Deletion of the 445-nucleotide (nt) 3'-untranslated region (3'-UTR) (L/++(+)-) or 527 nt of the 3'-coding region (3'-CR) (L/++-+) increased the stability of NF-L mRNA compared with the full-length (L/++(++)) transcript in neuronal (N2a and P19 cells) and 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 stability determinant was then localized to a 68-nt sequence that forms the junction between the 3'-CR and 3'-UTR of NF-L and is the binding site of a unique ribonucleoprotein complex (Cañete-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 determinants that regulate the stability of the NF-L mRNA.
Cañete-Soler
R
R
Division of Neuropathology, Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA. racansol@mail.med.upenn.edu
Schwartz
M L
ML
Hua
Y
Y
Schlaepfer
W W
WW
D000818:Animals; D002460:Cell Line; D051379:Mice; D016900:Neurofilament Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics*; D013752:Tetracycline / Q000494:pharmacology*; D015534:Trans-Activators / Q000378:metabolism*
D016900:Neurofilament Proteins; D012333:RNA, Messenger; D015534:Trans-Activators; C044882:neurofilament protein L; D013752:Tetracycline