Human breast cancer cells contain elevated levels and activity of the protein kinase, PKR.

PKR is a double-stranded (ds) RNA activated protein kinase whose expression is induced by interferon. Activated PKR phosphorylates its cellular substrate, eIF2, an essential initiation factor of translation. Prior evidence from a murine model system suggested that PKR may act as a tumor suppressor, but the evidence from human tumors is equivocal. To study PKR function in human breast cancer, PKR activity was measured in mammary carcinoma cell lines and nontransformed mammary epithelial cell lines. If PKR functioned as a tumor suppressor in this system, its activity would be higher in nontransformed cells than in carcinoma cells. On the contrary, PKR autophosphorylation and the phosphorylation of its substrate, the alpha-subunit of eIF2, is 7 - 40-fold higher in lysates prepared from breast carcinoma cell lines than in those from nontransformed epithelial cell lines. Correspondingly, a larger proportion of eIF2alpha is present in a phosphorylated state in carcinoma cell lines than in nontransformed cell lines. Protein synthesis is not inhibited by the high eIF2alpha phosphorylation in carcinoma cells, probably because they contain higher levels of eIF2B, the initiation factor that is inhibited by eIF2alpha phosphorylation. The dramatically lower PKR activity in nontransformed cell lines is partially due to lower PKR protein levels (2 - 4-fold) as well as to the presence of a PKR inhibitor. The nontransformed cells contain P58, a known cellular inhibitor of PKR that physically interacts with PKR and may be responsible for the low PKR activity in these cells. Taken together, these observations call into question the role of PKR as a tumor suppressor and suggest a positive regulatory role of PKR in growth control of breast cancer cells.

Termination sequence requirements vary among genes transcribed by RNA polymerase III.

RNA polymerase III (pol III) transcription generally terminates at a run of four or more thymidine (T) residues but some pol III genes contain runs of T residues that are not recognized as termination signals. Here, we investigate the terminal signal requirements that are operative in adenovirus virus-associated (VA) RNA genes. In the Xenopus 5 S RNA gene, efficient termination requires the T residues to be in a G+C-rich sequence context, but a run of five T residues in a G+C-rich context does not cause pol III termination when placed 30 nt downstream of the adenovirus-2 VA RNAI promoter in a VA-Tat chimeric gene. The failure of pol III to recognize this putative termination signal is not due to the chimeric nature of the gene or to the proximity of the signal to the promoter, but to its sequence context. Termination at the VA RNA gene site requires a T-rich sequence and is inhibited by the proximity of G residues, but is insensitive to the presence of A residues. The T-rich sequence need not be uninterrupted, however. In the VA RNA gene of the avian adenovirus, CELO, the first of two tandem termination signals contains an interrupted run of T residues, TTATT, which functions as a terminator with high (although not complete) efficiency. These findings, together with a survey of sequences neighboring the terminal site of other pol III genes, lead to the conclusion that pol III termination signals are more complex than hitherto recognized, and that sequence context requirements differ between members of the class 1 and class 2 families of pol III genes.

RNA binding and modulation of PKR activity.

PKR is an RNA-dependent protein kinase that is induced in mammalian cells by interferon treatment. It is present in a latent or inactive form in mammalian cells and is activated by very low concentrations of double-stranded (ds) RNA. Activated PKR phosphorylates eIF2, an essential initiation factor of protein synthesis, as well as other substrates including histone IIA, a 90-kDa protein from rabbit reticulocytes, the inhibitor, IkappaB, of the transcription factor, NF-kappaB, and the HIV-1 Tat protein. PKR interacts with several cellular and viral products and these interactions modulate its activation by dsRNA. Here we describe methods that are used to study the activation or inhibition of PKR by RNA modulators. Specifically, we detail (1) the purification of PKR from interferon-treated mammalian cells, (2) functional assays for PKR activation and inhibition in vitro, using purified enzyme or crude cell lysates, and (3) assays allowing evaluation of the binding of dsRNA and single-stranded RNA to PKR.

Translation of an uncapped mRNA involves scanning.

tat, an essential gene of human immunodeficiency virus, when placed under the control of the RNA polymerase III promoter from the adenovirus VA RNA1 gene, is transcribed into an uncapped and nonpolyadenylated mRNA. This VA-Tat RNA is translated to produce functional Tat protein in transfected mammalian cells (Gunnery, S., and Mathews, M. B. (1995) Mol. Cell. Biol. 15, 3597-3607). The presence of an upstream open reading frame (ORF) in VA-Tat RNA is inhibitory to the translation of the Tat ORF, suggesting that the RNA is scanned during translation even though it is uncapped. Because the effect of the upstream ORF is relatively small (about 2-fold), we sought more definitive evidence of scanning by introducing secondary structures of varying stabilities into the 5'-untranslated region of VA-Tat RNA. The results of transfection experiments showed that highly stable secondary structure was inhibitory to Tat synthesis, whereas structures of lower stability were not inhibitory, confirming that uncapped mRNA is subject to scanning. Furthermore, translation of the downstream ORF was reduced but not eliminated by mutations that caused the upstream ORF to overlap the Tat ORF. Extending the overlap of the two ORFs further decreased the translation of the downstream ORF. This observation implies that ribosomes reinitiate after termination, possibly after migrating in a 3' to 5' direction through the overlap region of the mRNA. Similar results were obtained with a capped polymerase II transcript, indicating that the translation of polymerase II and polymerase III transcripts occurs through similar mechanisms.

Functional mRNA can be generated by RNA polymerase III.

Eukaryotic cellular mRNA is believed to be synthesized exclusively by RNA polymerase II (pol II), whereas pol I produces long rRNAs and pol III produces 5S rRNA, tRNA, and other small RNAs. To determine whether this functional differentiation is obligatory, we examined the translational potential of an artificial pol III transcript. The coding region of the human immunodeficiency virus type 1 tat gene was placed under the control of a strong pol III promoter from the adenovirus type 2 VA RNAI gene. The resultant chimera, pVA-Tat, was transcribed accurately in vivo and in vitro and gave rise to Tat protein, which transactivated a human immunodeficiency virus-driven chloramphenicol acetyltransferase reporter construct in transfected HeLa cells. pol III-specific mutations down-regulated VA-Tat RNA production in vivo and in vitro and dramatically reduced chloramphenicol acetyltransferase transactivation. As expected for a pol III transcript, VA-Tat RNA was not detectably capped at its 5' end or polyadenylated at its 3' end, but, like mRNA, it was associated with polysomes in a salt-stable manner. Mutational analysis of a short open reading frame upstream of the Tat-coding sequence implicates scanning in the initiation of VA-Tat RNA translation despite the absence of a cap. In comparison with tat mRNA generated by pol II, VA-Tat RNA was present on smaller polysomes and was apparently translated less efficiently, which is consistent with a relatively low initiation rate. Evidently, human cells are capable of utilizing pol III transcripts as functional mRNAs, and neither a cap nor a poly(A) tail is essential for translation, although they may be stimulatory. These findings raise the possibility that some cellular mRNAs are made by pol I or pol III.

The nucleotide sequence of genes involved in the leucine biosynthetic pathway of Clostridium pasteurianum.

A 2.2 kb SphI/ClaI fragment of the Clostridium pasteurianum chromosome has previously been cloned and shown to complement leuB401 and leuC171 mutations in Escherichia coli. The nucleotide sequence of this fragment has been determined (2327 bp) and carries three open reading frames. The products of translation of these reading frames display significant homologies with the alpha-isopropylmalate isomerase subunit (leuD) gene of Salmonella typhimurium, the beta-isopropylmalate dehydrogenase (leuB) genes of several organisms, and the dihydroxyacid dehydrase (ilvD) gene of E. coli.

Tat-responsive region RNA of human immunodeficiency virus type 1 stimulates protein synthesis in vivo and in vitro: relationship between structure and function.

The Tat-responsive region (TAR) sequence is present at the 5' end of human immunodeficiency virus 1 mRNAs and as a cytoplasmic form of 58-66 nucleotides. TAR RNA blocks the activation and autophosphorylation of the double-stranded RNA-activated protein kinase in vitro. We show here that TAR RNA also prevents the double-stranded RNA-mediated inhibition of translation in a cell-free system. Mutagenic and structural analyses of TAR RNA indicate that a stem of at least 14 base pairs is required for this activity, whereas the loop and bulge required for transactivation by Tat are dispensable. Truncation of the RNA to 68 nucleotides results in the loss of translational rescue ability, suggesting that the short cytoplasmic TAR RNA produced by viral transcription in vivo may not have the capability to suppress activation of the kinase. However, because longer TAR transcripts stimulate expression in a transient assay in vivo, the TAR structure at the 5' end of viral mRNAs could still exert this function in cis.

Tat-responsive region RNA of human immunodeficiency virus 1 can prevent activation of the double-stranded-RNA-activated protein kinase.

Transcription from the human immunodeficiency virus type 1 promoter gives rise to short cytoplasmic transcripts of approximately 60 nucleotides as well as to longer mRNAs. These RNAs contain the Tat-responsive region sequence, which is capable of assuming a stem-loop structure and has been implicated in the regulation of both transcription and translation. It has been reported that Tat-responsive region RNA inhibits translation in vitro through activation of an interferon-induced protein kinase, the double-stranded-RNA-activated inhibitor, which phosphorylates eukaryotic initiation factor 2. We show that the activation property is due to double-stranded RNA that often contaminates RNA synthesized in vitro using bacteriophage RNA polymerases. After purification, high concentrations of Tat-responsive region RNA inhibit the activation of double-stranded RNA-activated inhibitor, suggesting that it may serve to protect human immunodeficiency virus type 1 infection from a cellular defense mechanism.

Regulation of Protein Synthesis in Plant Embryo by Protein Phosphorylation : I. Purification and Characterization of a cAMP-Independent Protein Kinase and Its Endogenous Substrate.

A cyclic AMP-independent protein kinase, which strongly inhibits in vitro protein synthesis, was purified to homogeneity from barley embryo by affinity and ion exchange chromatography. The M(r) of the purified enzyme is 95,000 with two nonidentical subunits of M(r) 58,000 and 39,000. The enzyme activity is not stimulated by cAMP, cGMP, or calmodulin. The endogenous phosphate acceptor of this kinase is a protein of M(r) 52,000, was isolated by purified protein kinase immobilized Sepharose column. Using antibodies raised against this protein kinase, the levels of the enzyme during embryogenesis and germination are determined. An inverse relationship has been observed between protein kinase level and rate of protein synthesis.

An inhibitor RNA of translation from barley embryo.

A small molecular weight RNA isolated from barley embryos inhibits specifically protein synthesis initiation. It does not bind to Oligo (dT)-Cellulose indicating that it is devoid of long poly(A) stretches. It is a single stranded RNA as it is sensitive to pancreatic RNase. This is the first report of such an RNA in a plant system.