Processing of 9E3 mRNA and Regulation of Its Stability in Normal and Rous Sarcoma Virus-Transformed Cells

Abstract

We studied the expression of 9E3 mRNA, which is known to be induced in chicken embryo fibroblasts by p60^v-src activity and by serum. In addition to full-length 9E3 mRNA, we identified several smaller RNAs that hybridized with 9E3 cDNA. One of these RNAs hybridized with a 5′ 9E3 cDNA probe but not with a 3′ cDNA probe. The other hybridized with a 3′ cDNA probe but lacked 5′ sequences, including the entire 9E3 coding region. Only the latter RNA was polyadenylylated, as determined by RNase H digestion in the presence of oligo(dT). The level of the small RNAs increased after treatment with cycloheximide and actinomycin D, indicating that the small RNAs were produced by processing of preexisting transcripts. The derivation of the small RNAs from 9E3 mRNA rather than from a related gene was confirmed by S1 nuclease analysis. The 3′ terminus of the 5′ RNA and the 5′ terminus of the 3′ RNA mapped to the same position, which suggested that the small RNAs were formed by endonucleolytic cleavage of 9E3 mRNA at a specific site in the 3′ noncoding region. We also found that the stability of 9E3 mRNA was increased after serum stimulation and was greater in Rous sarcoma virus-transformed than in uninfected cells. The relative amount of the small RNAs as compared with the full-length transcript was greatest under conditions in which the full-length transcript was least stable. These data suggest that site-specific endonucleolytic cleavage regulates the stability of 9E3 mRNA.