Assay of a ribonuclease that preferentially hydrolyses mRNAs containing cytokine-derived UA-rich instability sequences.
mRNA molecules encoding a number of inflammatory cytokines, as well as certain proto-oncogenes, contain a conserved UA-exclusive sequence in the 3' untranslated region that confers message instability in vivo. This sequence may comprise a critical regulatory element, governing the level of these mRNA molecules, and determining the efficiency with which they are translated. Through the use of a double-label RNAse assay, we have determined that lysates prepared from mouse macrophages selectively degrade mRNA molecules containing the 3' untranslated UA sequence found in the mRNA encoding human cachectin/TNF. The degree of instability is dependent upon the number of copies of inserted UA sequence present in the target mRNA molecule (a Xenopus beta-globin mRNA). mRNAs containing randomly generated UA sequences are more labile than unmodified globin mRNA, but less susceptible to degradation than mRNAs containing the authentic cachectin-derived sequence. mRNA molecules containing synthetic UG-exclusive sequences are normally stable or protected in vitro. The destruction of UA-containing mRNA is inhibited by random adenylate/uridilate copolymers, but not by guanylate/uridilate copolymers. Boiling or proteinase K treatment destroys the selective nucleolytic activity of macrophage lysates. We propose that the nuclease measured here may serve to regulate cellular levels of mRNA molecules encoding cachectin, other inflammatory cytokines, and certain proto-oncogene products.
Beutler
B
B
Howard Hughes Medical Institute, University of Texas Health Science Center, Dallas 75235.
Thompson
P
P
Keyes
J
J
Hagerty
K
K
Crawford
D
D
D000225:Adenine / Q000032:analysis*; D000818:Animals; D001483:Base Sequence; D001688:Biological Products / Q000235:genetics*; D016207:Cytokines; D051379:Mice; D008809:Mice, Inbred C3H; D000090063:Proto-Oncogene Mas; D012333:RNA, Messenger / Q000378:metabolism*; D012260:Ribonucleases / Q000378:metabolism*; D014529:Uridine / Q000032:analysis*; D014982:Xenopus laevis
D001688:Biological Products; D016207:Cytokines; C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D012333:RNA, Messenger; D012260:Ribonucleases; D000225:Adenine; D014529:Uridine