Human beta-globin mRNAs that harbor a nonsense codon are degraded in murine erythroid tissues to intermediates lacking regions of exon I or exons I and II that have a cap-like structure at the 5' termini.
Previous studies have demonstrated that nonsense codons within beta zero-thalassemic or in vitro-mutagenized human beta-globin transgenes result in the production of mRNAs that are degraded abnormally rapidly in the cytoplasm of murine erythroid cells. As a consequence, three RNA degradative intermediates are formed that lack sequences from either exon I or exons I and II. We show here that the intermediates, like the full-length mRNA from which they derive and the endogenous murine beta maj-globin mRNA, bind to the anticap monoclonal antibody H-20 in a way that is competed by the cap analogue m7G and eliminated by prior exposure to tobacco acid pyrophosphatase. Furthermore, the intermediates, like the two full-length mRNAs, are resistant to a 5'----3' exonuclease activity isolated from HeLa cell nuclei that degrades uncapped but not capped ribopolymers. Based on these observations, the intermediates appear to possess a structure that is indistinguishable from the cap at the 5' end of mRNA, i.e. a methylated nucleoside that is linked to the RNA by a 5'-5' phosphodiester bond. Detection of the intermediates during murine development was concomitant with detection of full-length thalassemic mRNA. Intermediate production appears to be influenced by RNA structure as indicated by the products that derive from a beta zero-thalassemic beta-globin transgene harboring a structural alteration (a 4 bp deletion) that was larger than any of those previously studied.
Lim
S K
SK
Department of Human Genetics, Roswell Park Cancer Institute, Buffalo, NY 14263.
Maquat
L E
LE
D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D003062:Codon; D015672:Erythroid Precursor Cells / Q000378:metabolism; D005091:Exons; D005092:Exonucleases / Q000494:pharmacology; D005914:Globins / Q000235:genetics*; D006801:Humans; D008745:Methylation; D008780:Methyltransferases; D051379:Mice; D008969:Molecular Sequence Data; D011755:Pyrophosphatases / Q000494:pharmacology; D012315:RNA Caps / Q000302:isolation & purification / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000187:drug effects / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D013154:Spleen / Q000378:metabolism; D013789:Thalassemia / Q000235:genetics*
D000911:Antibodies, Monoclonal; D003062:Codon; D012315:RNA Caps; D012333:RNA, Messenger; D005914:Globins; D008780:Methyltransferases; D005092:Exonucleases; D011755:Pyrophosphatases