Abstract
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.
Highlights
Altering mRNA stability is a universal post-transcriptional mechanism for modulating gene expression during growth, development and differentiation
The method has enabled the identification of stability determinants in the 3Ј-CR and 3Ј-UTR of the NF-L mRNA and have localized a key determinant to a 68-nt sequence at the junction of the 3Ј-CR and 3Ј-UTR
Short term (6 h) losses of NF-L mRNAs from wild-type and mutant transgenes in P19 cells showed intermediate levels of decline, supporting the view that decreases in the rate of loss of NF-L/actin mRNA over time may be due to increasing admixtures of mRNA from the endogenous NF-L gene
Summary
Altering mRNA stability is a universal post-transcriptional mechanism for modulating gene expression during growth, development and differentiation. To monitor the tetracycline-responsive promoter system for both activation and inactivation of the test gene, the luciferase reporter gene was stably transfected into cell lines containing the autoinducible tTA transactivator expression vector (pUHD15.1M).
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
