Our previous studies of transgenic mice have demonstrated that nonsense codons within beta-thalassemic or in vitro-mutagenized human beta-globin transgenes result in the production of beta-globin mRNAs that are degraded abnormally rapidly in erythroid cells. Three RNA degradative intermediates are formed that are polyadenylated but lack sequences from either exon 1 or exons 1 and 2. The intermediates appear to be capped at the 5' end since, like the full-length mRNA from which they derive and the endogenous mouse beta-globin mRNA, (i) they bind to a monoclonal cap antibody in a way that is competed by the cap analogue m7G and eliminated by prior exposure to tobacco acid pyrophosphatase, and (ii) are resistant to a 5'- to-3' exonuclease activity isolated from HeLa cell nuclei that degrades uncapped but not capped ribopolymers. Our ability to characterize further the degradative intermediates using the transgenic animals is limited by (i) the low abundance of mRNA that derives from the human beta-globin transgene relative to the abundance of mRNA that derives from the endogenous mouse beta-globin genes, especially given the high degree of sequence similarity between the mRNAs, (ii) the inability to harvest large quantities of transcriptionally active erythroid cells, and (iii) the presence of nucleases that hamper erythroid cell fractionation studies. To continue these studies, a normal, a thalassemic and an in vitro- reverted thalassemic beta-globin allele have been stably integrated into the genome of cultured mouse erythroleukemia (MEL) cells using a vector that directs a high level of beta-globin gene expression in a copy number- dependent and integration site-independent manner. Upon induction of globin gene expression using dimethylsulfoxide, the cultured cells that express the thalassemic allele are characterized by an abnormally low level of full-length beta-globin mRNA as well as three shorter beta-globin RNAs that are similar in size to the thalassemic mRNA decay products of the transgenic animals. In contrast, the cultured cells that express the reverted thalassemic allele, which harbors the thalassemic deletion and a compensating insertion, are characterized by normal beta-globin RNA metabolism.
We aim to utilize these and additional MEL cell transfectants that will be constructed to elucidate the pathway of nonsense codon- mediated RNA decay. To this end, the presumed precursor-product and structural relationships between full-length mRNA and the decay products will be analyzed. If like S. cerevisiae, the nonsense codon-mediated decay pathway will derive from a pathway of normal mRNA decay and neither pathway will be unique to beta-globin mRNA.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM052822-01
Application #
2191988
Study Section
Molecular Biology Study Section (MBY)
Project Start
1995-08-01
Project End
1999-07-31
Budget Start
1995-08-01
Budget End
1996-07-31
Support Year
1
Fiscal Year
1995
Total Cost
Indirect Cost
Name
Roswell Park Cancer Institute Corp
Department
Type
DUNS #
City
Buffalo
State
NY
Country
United States
Zip Code
14263