Control of c-myc mRNA half-life in vitro by a protein capable of binding to a coding region stability determinant.
Polysome-associated c-myc mRNA is degraded relatively rapidly in cells and in an in vitro mRNA decay system containing extracts from cultured mammalian cells. Using this system, a competition/screening assay was devised to search for factors that bind to specific regions of polysome-associated c-myc mRNA and thereby alter its half-life. mRNA stability was first assayed in reactions containing exogenous competitor RNAs corresponding to portions of c-myc mRNA itself. The addition of a 182-nucleotide sense strand fragment from the carboxy-terminal portion of the c-myc-coding region destabilized c-myc mRNA by at least eightfold. This RNA fragment had no effect on the stability of other mRNAs tested. Moreover, c-myc mRNA was not destabilized in reactions containing unrelated competitor RNAs or sense strand RNA from the c-myc 5' region. Polysome-associated globin mRNA containing the c-myc-coding region segment in-frame was also destabilized in vitro by the 182-nucleotide RNA. As determined by UV-cross-linking experiments, the 182-nucleotide RNA fragment was recognized by and bound to an approximately 75-kD polysome-associated protein. On the basis of these data plus Northern blotting analyses of c-myc mRNA decay products, we suggest that the approximately 75-kD protein is normally bound to a c-myc-coding region determinant and protects that region of the mRNA from endonuclease attack. Possible links between the protective protein, translation, ribosome pausing, and c-myc mRNA turnover are discussed.
Bernstein
P L
PL
McArdle Laboratory for Cancer Research, University of Wisconsin, Madison 53706.
Herrick
D J
DJ
Prokipcak
R D
RD
Ross
J
J
D001483:Base Sequence; D002678:Chimera; D004591:Electrophoresis, Polyacrylamide Gel; D016259:Genes, myc; D005914:Globins / Q000235:genetics; D006207:Half-Life; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids; D011132:Polyribosomes; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects; D014407:Tumor Cells, Cultured; D014466:Ultraviolet Rays
D012333:RNA, Messenger; D005914:Globins