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An amino acid starvation-induced mRNA, increased up to 4-fold in the absence of amino acids, was identified previously as rat 60 S subunit ribosomal protein L17. The data presented here demonstrate that among ribosomal proteins, L17, as well as the smaller subunit ribosomal protein S25, are uniquely regulated by amino acid deprivation of cells; the increase in L17 and S25 mRNA content in response to substrate starvation is not shared by the 11 other ribosomal proteins tested. When rat Fao hepatoma cells were incubated in the presence of actinomycin D, the L17 mRNA level decayed below the basal level, regardless of medium amino acid content, and nuclear run-off assays confirmed that nutrient starvation leads to enhanced transcription of the L17 ribosomal protein gene. Likewise, the induction of S25 mRNA also was prevented in the presence of actinomycin D. Furthermore, the induction of L17 and S25 mRNA content was blocked by cycloheximide, demonstrating the requirement for a newly synthesized protein in the signaling pathway. Northern analysis with RNA isolated from cytoplasmic, polysomal, and nuclear enriched fractions indicated that the starvation-dependent increase in the S25 and L17 mRNAs is retained within the nucleus and not is available for translation. Amino acid refeeding of the cells caused the translocation of the stored nuclear mRNAs to the polysomal fraction.

Amino acid deprivation of rat hepatoma cells induced the levels of a 612-base pair mRNA termed ASI (Shay, N. F., Nick, H. S., and Kilberg, M. S. (1990) J. Biol. Chem. 265, 17844-17848). The ASI mRNA was present at levels equal to or greater than actin in every rat tissue tested. The corresponding full-length cDNA was cloned, and the present report demonstrates that the deduced 184-residue amino acid sequence shares greater than 30% identity to a number of bacterial and chloroplast L22 ribosomal proteins, including those from Escherichia coli and Halobacterium halobium. A monospecific anti-peptide antibody was produced that upon immunochemical analysis of subcellular fractions of rat liver recognized a band in the microsomal fraction and, more specifically, reacted with a single polypeptide in the ribosomal large subunit fraction. The antibody did not react with any proteins of the mitochondrial large subunit, but did recognize a protein in human liver homogenate at the same relative mobility (23 kDa) as that observed for rat liver.

Using the combination of a subtracted library and differential hybridization, a 409-base pair cDNA was identified that corresponds to a mRNA that is induced 2-3-fold when rat Fao hepatoma cells are subjected to amino acid starvation for 12 h. While this mRNA species was induced during starvation, others such as beta-actin, Cu-Zn superoxide dismutase, glyceraldehyde-3-P, and histone H4 were decreased in abundance to 25-50% of their original levels. The induction of the amino acid starvation-induced (ASI) mRNA was repressed when starved cells were returned to a medium supplemented with amino acids. Tissue distribution analysis showed the ASI mRNA, approximately 650 base pairs in length, to be present in every rat tissue tested. The cDNA clone has been sequenced and appears to correspond to the 3'-most end of the mRNA. The cDNA sequence includes the poly(A) tail, two potential polyadenylation signal sequences, and an open reading frame that we presume to be a portion of the coding sequence. The ASI cDNA will be used to investigate the molecular mechanisms for amino acid-dependent regulation of protein expression by mammalian cells.