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.

Key Points Cytokine-activated NK cells display distinct gene expression programs in response to cytokine withdrawal. IL-15 sustains antitumor functions of NK cells through mTOR-governed metabolic processes.

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Biofilms similar to those present in water distribution pipes of anthropogenic aquatic systems were simulated in a rotating annular reactor using a non-Legionella community consisting of Aeromonas hydrophila, Escherichia coli, Flavobacterium breve and Pseudomonas aeruginosa. The impact of this community and Acanthamoeba castellanii on the replication of Legionella pneumophila was investigated. Despite the presence of 10(7) non-Legionella bacteria, culture and real-time polymerase chain reaction (PCR) results clearly showed that biofilm-associated Legionella bacteria only increased after intracellular replication in A. castellanii. Fluorescent in situ hybridization (FISH) staining of biofilm samples revealed that 48 h after addition of amoebae to the reactor, the amoeba population was lysing and replicated Legionella bacteria were released into the bulk water. This study demonstrated that amoebae like A. castellanii can play a crucial role in the increase and spread of L. pneumophila in anthropogenic aquatic systems and thus in the occurrence of Legionnaires' disease.

The integrated stress response (ISR) is an essential stress-support pathway increasingly recognized as a determinant of tumorigenesis. Here we demonstrate that ISR is pivotal in lung adenocarcinoma (LUAD) development, the most common histological type of lung cancer and a leading cause of cancer death worldwide. Increased phosphorylation of the translation initiation factor eIF2 (p-eIF2α), the focal point of ISR, is related to invasiveness, increased growth, and poor outcome in 928 LUAD patients. Dissection of ISR mechanisms in KRAS-driven lung tumorigenesis in mice demonstrated that p-eIF2α causes the translational repression of dual specificity phosphatase 6 (DUSP6), resulting in increased phosphorylation of the extracellular signal-regulated kinase (p-ERK). Treatments with ISR inhibitors, including a memory-enhancing drug with limited toxicity, provides a suitable therapeutic option for KRAS-driven lung cancer insofar as they substantially reduce tumor growth and prolong mouse survival. Our data provide a rationale for the implementation of ISR-based regimens in LUAD treatment.

Estrogen receptor alpha (ERα) activity is associated with increased cancer cell proliferation. Studies aiming to understand the impact of ERα on cancer‐associated phenotypes have largely been limited to its transcriptional activity. Herein, we demonstrate that ERα coordinates its transcriptional output with selective modulation of mRNA translation. Importantly, translational perturbations caused by depletion of ERα largely manifest as “translational offsetting” of the transcriptome, whereby amounts of translated mRNAs and corresponding protein levels are maintained constant despite changes in mRNA abundance. Transcripts whose levels, but not polysome association, are reduced following ERα depletion lack features which limit translation efficiency including structured 5′UTRs and miRNA target sites. In contrast, mRNAs induced upon ERα depletion whose polysome association remains unaltered are enriched in codons requiring U34‐modified tRNAs for efficient decoding. Consistently, ERα regulates levels of U34‐modifying enzymes and thereby controls levels of U34‐modified tRNAs. These findings unravel a hitherto unprecedented mechanism of ERα‐dependent orchestration of transcriptional and translational programs that may be a pervasive mechanism of proteome maintenance in hormone‐dependent cancers.

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Polysome-profiling is commonly used to study translatomes and applies laborious extraction of efficiently translated mRNA (associated with >3 ribosomes) from a large volume across many fractions. This property makes polysome-profiling inconvenient for larger experimental designs or samples with low RNA amounts. To address this, we optimized a non-linear sucrose gradient which reproducibly enriches for efficiently translated mRNA in only one or two fractions, thereby reducing sample handling 5–10-fold. The technique generates polysome-associated RNA with a quality reflecting the starting material and, when coupled with smart-seq2 single-cell RNA sequencing, translatomes in small tissues from biobanks can be obtained. Translatomes acquired using optimized non-linear gradients resemble those obtained with the standard approach employing linear gradients. Polysome-profiling using optimized non-linear gradients in serum starved HCT-116 cells with or without p53 showed that p53 status associates with changes in mRNA abundance and translational efficiency leading to changes in protein levels. Moreover, p53 status also induced translational buffering whereby changes in mRNA levels are buffered at the level of mRNA translation. Thus, here we present a polysome-profiling technique applicable to large study designs, primary cells and frozen tissue samples such as those collected in biobanks.