No abstract

SUMMARY Integrated Stress Response is a homeostatic mechanism induced by endoplasmic reticulum (ER) stress. In acute/transient ER stress, decreased global protein synthesis and increased uORF mRNA translation are followed by normalization of protein synthesis. Here, we report a dramatically different response during chronic ER stress. This chronic ISR program is characterized by persistently elevated uORF mRNA translation and concurrent gene expression reprogramming, which permits simultaneous stress sensing and proteostasis. The program includes PERK-dependent switching to an eIF3-dependent translation initiation mechanism resulting in partial but not complete translational recovery, which, together with transcriptional reprogramming, selectively bolsters expression of proteins with ER functions. Coordination of transcriptional and translational reprogramming prevents ER dysfunction and inhibits “foamy cell” development, thus establishing a molecular basis for understanding human diseases associated with ER dysfunction.

A recombinant vaccinia virus that directs the synthesis of bacteriophage T7 RNA polymerase provides the basis for the expression of genes that are regulated by T7 promoters in mammalian cells. The T7 transcripts, which account for as much as 30% of the total cytoplasmic RNA at 24 hr after infection, are largely uncapped. To improve the translatability of the uncapped RNA, the encephalomyocarditis virus (EMCV) untranslated region (UTR) was inserted between the T7 promoter and the chloramphenicol acetyltransferase (CAT) gene. Experiments with a reticulocyte extract demonstrated that the EMCV UTR conferred efficient and cap-independent translatability to CAT RNA synthesized in vitro by T7 RNA polymerase. In cells infected with recombinant vaccinia viruses containing the T7 promoter-regulated CAT gene, the EMCV UTR increased the amount of CAT RNA on polyribosomes. The polyribosome-derived CAT RNA, which contained the EMCV UTR, was translated in vitro in a capindependent fashion as well. Use of the EMCV UTR significantly enhanced the vaccinia/T7 hybrid expression system as it resulted in a 4-to 7-fold increase in total CAT activity. A further =2-fold improvement was achieved by incubating the cells in hypertonic medium, which favors the translation of uncapped picornavirus RNA over cellular mRNAs. With this newly modified expression system, CAT was the predominant protein synthesized by infected cells and within 24 hr accounted for >10% of the total cell protein.A eukaryotic expression system based on a recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase in the cytoplasm of infected mammalian cells was described previously (1-3). The target gene for the bacteriophage RNA polymerase, flanked by T7 promoter and termination sequences, was introduced into infected cells either by transfection of a recombinant plasmid or by infection with a second recombinant vaccinia virus. Through the use of transfection or infection protocols, it was found that T7 lacZ transcripts comprise 10% or 30%6, respectively, of the total cytoplasmic RNA at 24 hr after infection. The T7 transcripts were initiated correctly, but only 5-10% contained 5'-terminal cap structures, providing an explanation for the discrepancy between the major amount ofRNA made and the moderate amount of protein expressed. The more efficient capping of vaccinia virus mRNA, compared to T7 RNA, may be due to association of the viral RNA guanylyltransferase with the viral RNA polymerase (4). In addition, the 5' end stem-loop structure of the 17 transcripts, which was found to be crucial for its stability (3), might interfere with capping as well as with ribosome binding and scanning.To improve the translatability of the uncapped 17 tran-

Down syndrome, the phenotypic expression of human trisomy 21, is presumed to result from a 1.5-fold increase in the expression of the genes on human chromosome 21. As an approach to the development of an animal model for Down syndrome, several strains of transgenic mice that carry the human Cu/Zn-superoxide dismutase gene have been prepared. These animals express the transgene in a manner similar to that of humans, with 0.9-and 0.7-kilobase transcripts in a 1:4 ratio, and synthesize the human enzyme in an active form capable offorming human-mouse enzyme heterodimers. Cu/Znsuperoxide dismutase activity is increased from 1.6-to 6.0-fold in the brains of four transgenic strains and to an equal or lesser extent in several other tissues. These animals provide a unique system for studying the consequences of increased dosage of the Cu/Zn-superoxide dismutase gene in Down syndrome and the role of this enzyme in a variety of other pathological processes.