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.
Background: Protein synthesis control is important for -cell fate during ER stress. Results: Increased protein synthesis during chronic ER stress in -cells involves the transcriptional induction of an amino acid transporter network. Conclusion: Increased amino acid uptake in -cells during ER stress promotes apoptosis. Significance: Induced expression of a network of amino acid transporters in islets can contribute to chronic ER stress-induced diabetes.
Plasticity of neoplasia, whereby cancer cells attain stem-cell-like properties, is required for disease progression and represents a major therapeutic challenge. We report that in breast cancer cells NANOG, SNAIL and NODAL transcripts manifest multiple isoforms characterized by different 5' Untranslated Regions (5'UTRs), whereby translation of a subset of these isoforms is stimulated under hypoxia. The accumulation of the corresponding proteins induces plasticity and "fate-switching" toward stem cell-like phenotypes. Mechanistically, we observe that mTOR inhibitors and chemotherapeutics induce translational activation of a subset of NANOG, SNAIL and NODAL mRNA isoforms akin to hypoxia, engendering stem-celllike phenotypes. These effects are overcome with drugs that antagonize translational reprogramming caused by eIF2α phosphorylation (e.g. ISRIB), suggesting that the Integrated Stress Response drives breast cancer plasticity. Collectively, our findings reveal a mechanism of induction of plasticity of breast cancer cells and provide a molecular basis for therapeutic strategies aimed at overcoming drug resistance and abrogating metastasis.
cis-acting structures in positive-strand [(ϩ)-strand] RNA virus genomes that function as signals for genome translation, transcription, and replication are potential sites for antiviral drug design. The fact that replication signals from evolutionarily divergent coronaviruses can be exchanged, for example, from the 3Ј untranslated region (UTR) of the (group 2a) bovine coronavirus (BCoV) to the (group 2a) mouse hepatitis coronavirus (MHV) (21)(22) or from the (group 2b) severe acute respiratory syndrome (SARS) coronavirus (SCoV) to MHV (15), would suggest that designed therapeutic agents against common signals might be broadly effective. Several studies have identified cis-replication structures in the 3Ј UTR of MHV, heretofore recognized as the group 2 type species, and in the 3Ј UTR of BCoV that are likely involved in the initial steps of genome translation and initiation of negativestrand [(Ϫ)-strand] synthesis. These elements, identified in either a helper virus-dependent defective interfering (DI) RNA of MHV or BCoV or by reverse genetics in the fulllength MHV genome, are the following: (i) the 3Ј poly(A) tail (26, 34), (ii) the 3Ј-terminal 55 nucleotides (nt) of the 3Ј UTR (14,23,26,42), and (iii) an upstream bulged stem-loop (SL) and associated hairpin pseudoknot (13,(21)(22)36). Curiously, an ϳ140-nt hypervariable region in the 3Ј UTR that comprises part of a 3Ј-proximal bulged stem-loop and harbors a coronavirus-universal octamer sequence (GGAAGAGC) is not required for MHV replication in cell culture but plays a role in MHV pathogenesis (14).cis-replication structures have also been identified in the 5Ј-proximal region of group 2 coronaviruses, but few studies have characterized their common signaling features. In BCoV, the very 5Ј end of the genome exhibits a puzzling hypervariability during virus replication in that 5Ј extensions of up to 9 nt and varying sequences within the first 14 nt are found, but the full significance of this in virus replication is unknown (19). The higher-order RNA structures within the BCoV 5Ј UTR were initially predicted by the Tinoco algorithm (35) and more recently by the mfold algorithm of Zuker (31, 41). They have been characterized as SLs I to IV (Fig. 1B) and were shown to be consistent with enzyme structure probing analyses (5-6, 32-33). More recent characterizations based on comparisons with MHV have shown SLI to be comprised of two smaller stem-loops, named SL1 and SL2 (Fig. 1B) (24)(25)(27)(28). In the context of the DI RNA in BCoV-infected cells, it was not feasible to determine whether SLI or SLII is a bona fide cisreplication element, since SLI resides within the 65-nt leader and SLII resides largely within the leader, and transfected DI RNAs containing an experimentally mutated leader rapidly acquired the leader sequence of the helper virus genome in a process known as "leader switching" (5-6, 29). SLII, furthermore, harbors the leader-associated transcription regulatory core sequence (UCUAAAC) responsible for the RNA-depen-* Corresponding author. Mailing addres...
Higher-order RNA structures in the 5= untranslated regions (UTRs) of the mouse hepatitis coronavirus (MHV) and bovine coronavirus (BCoV), separate species in the betacoronavirus genus, appear to be largely conserved despite an ϳ36% nucleotide sequence divergence. In a previous study, each of three 5=-end-proximal cis-acting stem-loop domains in the BCoV genome, I/II, III, and IV, yielded near-wild-type (wt) MHV phenotypes when used by reverse genetics to replace its counterpart in the MHV genome. Replacement with the BCoV 32-nucleotide (nt) inter-stem-loop fourth domain between stemloops III and IV, however, required blind cell passaging for virus recovery. Here, we describe suppressor mutations within the transplanted BCoV 32-nt domain that along with appearance of potential base pairings identify an RNA-RNA interaction between this domain and a 32-nt region ϳ200 nt downstream within the nonstructural protein 1 (Nsp1)-coding region. Mfold and phylogenetic covariation patterns among similarly grouped betacoronaviruses support this interaction, as does cotransplantation of the BCoV 5= UTR and its downstream base-pairing domain. Interestingly, cotransplantation of the BCoV 5= UTR and BCoV Nsp1 coding region directly yielded an MHV wt-like phenotype, which demonstrates a cognate interaction between these two BCoV regions, which in the MHV genome act in a fully interspecies-compliant manner. Surprisingly, the 30-nt inter-stem-loop domain in the MHV genome can be deleted and viral progeny, although debilitated, are still produced. These results together identify a previously undescribed long-range RNA-RNA interaction between the 5= UTR and Nsp1 coding region in MHV-like and BCoV-like betacoronaviruses that is cis acting for viral fitness but is not absolutely required for viral replication in cell culture. In positive-strand RNA viruses that replicate in the cytoplasm, genomic 5=-end-proximal RNA structures carry out several functions required for virus reproduction. In coronaviruses, these are thought to include (i) translation initiation, commonly presumed to occur by a canonical cap-dependent 5=-terminal ribosomal entry mechanism, to synthesize the replicase enzymes from open reading frame 1 (13, 14, 31); (ii) signaling an RNA-dependent RNA polymerase template switch during minus-strand synthesis at a heptameric transcription-regulatory sequence (UCUAAAC in the case of mouse hepatitis coronavirus [MHV] and bovine coronavirus [BCoV]) (Fig. 1A and B) for placement of a common leader on subgenomic mRNAs (sgmRNAs) (43,48,49,55); (iii) encoding signals on the 3= end of minus-strand genomic RNA (the antigenome) for initiating synthesis of plus-strand genomic mRNAs and sgmRNAs (9, 43, 48, 55); (iv) possibly harboring signals that act in trans to initiate synthesis of nascent plus-strand genomes (46); (v) possibly directly influencing initiation of minus-strand synthesis at the 3= end of the genome (33); and (vi) harboring a genome packaging signal (10,18). A mechanistic understanding of these events may aid in the devel...
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