Degradation of unstable interleukin-1 alpha mRNA in a rabbit reticulocyte cell-free system. Localization of an instability determinant to a cluster of AUUUA motifs.
Labeled transcripts of interleukin-1 alpha (IL-1 alpha) cDNA were rapidly degraded in incubations with rabbit reticulocyte lysate (RRL). In contrast, a transcript of superoxide dismutase cDNA was stable in control incubations. A transcript of the 3'-untranslated region (UTR) of IL-1 alpha was rapidly degraded while that of the 5'-UTR and coding region was stable. This degradative activity was present in the post-ribosomal supernatant. Degradation of the 3'-UTR transcript was inhibited by the addition of a large excess of an 80-base RNA containing four AUUUA repeats, but not by the same RNA without such repeats. This suggested that AUUUA motifs were responsible for the instability of the 3'-UTR transcript. The 80-base RNA did not act as a competitive substrate for a nuclease since it was not degraded. Partial transcripts of IL-1 alpha 3'-UTR were incubated with RRL to localize instability determinants. Transcripts containing at least three clustered AUUUA motifs were rapidly degraded, while transcripts containing four scattered AUUUA motifs were stable. To study the mechanism of RNA degradation, the RRL was passed through an affinity column that retained AUUUA-binding proteins. The flow-through or the fraction eluted from such a column were inactive, but the two fractions together degraded the 3'-UTR transcript. This indicated that proteins bound by the affinity column did not have nuclease activity but targeted this RNA for degradation.
Gorospe
M
M
Department of Biological Sciences, State University of New York, Albany 12222.
Baglioni
C
C
D000818:Animals; D001483:Base Sequence; D002474:Cell-Free System; D017931:DNA Primers; D018076:DNA, Complementary / Q000378:metabolism; D005033:Ethylmaleimide / Q000494:pharmacology; D007375:Interleukin-1 / Q000096:biosynthesis*; D007700:Kinetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D011817:Rabbits; D012091:Repetitive Sequences, Nucleic Acid; D012156:Reticulocytes / Q000378:metabolism*; D014158:Transcription, Genetic
D017931:DNA Primers; D018076:DNA, Complementary; D007375:Interleukin-1; D012333:RNA, Messenger; D005033:Ethylmaleimide