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Ternary complex (TC) and eIF4F complex assembly are the two major rate-limiting steps in translation initiation regulated by eIF2α phosphorylation and the mTOR/4E-BP pathway, respectively. How TC and eIF4F assembly are coordinated, however, remains largely unknown. We show that mTOR suppresses translation of mRNAs activated under short-term stress wherein TC recycling is attenuated by eIF2α phosphorylation. During acute nutrient or growth factor stimulation, mTORC1 induces eIF2β phosphorylation and recruitment of NCK1 to eIF2, decreases eIF2α phosphorylation and bolsters TC recycling. Accordingly, eIF2β mediates the effect of mTORC1 on protein synthesis and proliferation. In addition, we demonstrate a formerly undocumented role for CK2 in regulation of translation initiation, whereby CK2 stimulates phosphorylation of eIF2β and simultaneously bolsters eIF4F complex assembly via the mTORC1/4E-BP pathway. These findings imply a previously unrecognized mode of translation regulation, whereby mTORC1 and CK2 coordinate TC and eIF4F complex assembly to stimulate cell proliferation. Ternary complex (TC) and eIF4F complex assembly are rate-limiting steps in translation initiation that are regulated by eIF2α phosphorylation and the mTOR/4E-BP pathway. Here the authors show that the protein kinases mTORC1 and CK2 coordinate TC and eIF4F complex assembly through eIF2β to stimulate cell proliferation.

The protozoan parasite Leishmania donovani (L. donovani) causes visceral leishmaniasis, a chronic infection which is fatal when untreated. Herein, we investigated whether in addition to altering transcription, L. donovani modulates host mRNA translation to establish a successful infection. Polysome-profiling revealed that one third of protein-coding mRNAs expressed in primary mouse macrophages are differentially translated upon infection with L. donovani promastigotes or amastigotes. Gene ontology analysis identified key biological processes enriched for translationally regulated mRNAs and were predicted to be either activated (e.g. chromatin remodeling and RNA metabolism) or inhibited (e.g. intracellular trafficking and antigen presentation) upon infection. Mechanistic in silico and biochemical analyses showed selective activation mTOR- and eIF4A-dependent mRNA translation, including transcripts encoding central regulators of mRNA turnover and inflammation (i.e. PABPC1, EIF2AK2, and TGF-β). L. donovani survival within macrophages was favored under mTOR inhibition but was dampened by pharmacological blockade of eIF4A. Overall, this study uncovers a vast yet selective reprogramming of the host cell translational landscape early during L. donovani infection, and suggests that some of these changes are involved in host defense mechanisms while others are part of parasite-driven survival strategies. Further in vitro and in vivo investigation will shed light on the contribution of mTOR- and eIF4A-dependent translational programs to the outcome of visceral leishmaniasis.

Neuroblastic tumors include the neuroblastomas, ganglioneuroblastomas, and ganglioneuromas. Clinical behavior of these developmental malignancies varies from regression to aggressive growth with metastatic dissemination. Several clinical, histological, genetic, and biological features are associated with this diversity of clinical presentations. The calcium-sensing receptor (CaSR) is a G-protein coupled receptor with a key role in calcium homeostasis. We have previously reported that it is expressed in benign, differentiated neuroblastic tumors, but silenced by genetic and epigenetic events in unfavorable neuroblastomas. We have now analyzed three functionally relevant polymorphisms clustered at the signal transduction region of the CaSR (rs1801725, rs1042636 and rs1801726) to assess if genetic variants producing a less active receptor are associated with more aggressive disease course. Polymorphisms were analyzed in DNA samples from 65 patients using specific Taqman Genotyping Assays. Mildly inactivating variant rs1801725 was associated with clinical stage 4 (P = 0.002) and the histological subgroup of undifferentiated neuroblastomas (P = 0.046). Patients harboring this polymorphism had significantly lower overall (P = 0.022) and event-free survival (P = 0.01) rates than those who were homozygous for the most common allele among Caucasians. However, this single locus genotype was not independently associated with outcome in multivariate analyses. Conversely, the tri-locus haplotype TAC was independently associated with an increased risk of death in the entire cohort (Hazard Ratio = 2.45; 95% Confidence Interval [1.14-5.29]; P = 0.022) and also in patients diagnosed with neuroblastomas (Hazard Ratio = 2.74; 95% Confidence Interval [1.20-6.25]; P = 0.016). The TAC haplotype includes the moderately inactivating variant rs1801725 and absence of the gain-of-function rs1042636 polymorphism. Thus, its association with metastatic disease and poor outcome would add to our previous data and further support that inactivation of the CaSR gene is a mechanism associated with neuroblastoma malignant behavior.

Tumor-associated macrophages (TAMs) continuously fine tune their immune modulatory properties, but how gene expression programs coordinate this immune cell plasticity is largely unknown. Selective mRNA translation, controlled by MNK1/MNK2 and mTOR pathways impinging on eIF4E, facilitates reshaping of proteomes without changes in abundance of corresponding mRNAs. Using polysome profiling developed for small samples we show that, during tumor growth, gene expression in TAMs is predominately modulated via mRNA-selective changes in translational efficiencies. These alterations in gene expression paralleled accumulation of antiinflammatory macrophages with augmented phosphorylation of eIF4E, a target of the MNK1 and MNK2 kinases, known to selectively modulate mRNA translation. Furthermore, suppression of theMNK2, but not the mTOR signaling pathway, reprogrammed antiinflammatory macrophages toward a proinflammatory phenotype with the ability to activate CD8⁺ T cells. Thus, selective changes of mRNA translation depending on MNK2 signaling represents a key node regulating macrophage antiinflammatory functions.

Abstract Rationale Chronic obstructive pulmonary disease is an independent risk factor for lung cancer, but the underlying molecular mechanisms are unknown. We hypothesized that lung stromal cells activate pathological gene expression programs that support oncogenesis. Objectives To identify molecular mechanisms operating in the lung stroma that support the development of lung cancer. Methods The study included subjects with and without lung cancer across a spectrum of lung-function values. We conducted a multiomics analysis of nonmalignant lung tissue to quantify the transcriptome, translatome, and proteome. Measurements and Main Results Cancer-associated gene expression changes predominantly manifested as alterations in the efficiency of mRNA translation modulating protein levels in the absence of corresponding changes in mRNA levels. The molecular mechanisms that drove these cancer-associated translation programs differed based on lung function. In subjects with normal to mildly impaired lung function, the mammalian target of rapamycin (mTOR) pathway served as an upstream driver, whereas in subjects with severe airflow obstruction, pathways downstream of pathological extracellular matrix emerged. Consistent with a role during cancer initiation, both the mTOR and extracellular matrix gene expression programs paralleled the activation of previously identified procancer secretomes. Furthermore, an in situ examination of lung tissue showed that stromal fibroblasts expressed cancer-associated proteins from two procancer secretomes: one that included IL-6 (in cases of mild or no airflow obstruction), and one that included BMP1 (in cases of severe airflow obstruction). Conclusions Two distinct stromal gene expression programs that promote cancer initiation are activated in patients with lung cancer depending on lung function. Our work has implications both for screening strategies and for personalized approaches to cancer treatment.

The major purpose of the present study was to quantify correctly spliced CFTR transcripts in human nasal epithelial cells from cystic fibrosis (CF) patients carrying the splicing mutations c.580-1G>T (712-1G>T) and c.2657+5G>A (2789+5G>A) and to assess the applicability of this model in CFTR therapeutic approaches. We performed the relative quantification of CFTR mRNA by reverse transcription quantitative PCR (RT-qPCR) of these splicing mutations in four groups (wild type, CF-F508del controls, CF patients and CF carriers) of individuals. In addition, in vitro assays using minigene constructs were performed to evaluate the effect of a new CF complex allele c.[2657+5G>A; 2562T>G]. Ex vivo qPCR data show that the primary consequence of both mutations at the RNA level is the skipping of their neighboring exon (6 and 16, respectively). The CFTR minigenes results mimicked the ex vivo data, as exon 16 skipping is the main aberrant transcript, and the correctly spliced transcript level was observed in a similar proportion when the c.2657+5G>A mutation is present. In summary, we provide evidence that ex vivo quantitative transcripts analysis using RT/qPCR is a robust technology that could be useful for measuring the efficacy of therapeutic approaches that attempt to achieve an increase in CFTR gene expression.

Abstract Rationale Chronic obstructive pulmonary disease (COPD) is an independent risk factor for lung cancer, but the underlying molecular mechanisms are unknown. We hypothesized that stromal cells comprising the COPD lung contain pathological gene expression programs supporting oncogenesis. Objectives To identify molecular mechanisms operating in the lung stroma that support the development of lung cancer. Methods Study subjects included patients with COPD with (n = 30) or without (n = 30) lung cancer across a spectrum of COPD severity. We conducted multi-omics analysis of nonmalignant lung tissue to quantify the transcriptome, translatome, and proteome. Results Cancer-associated gene expression changes predominantly manifested as alterations in the efficiency of mRNA translation impacting protein levels in the absence of corresponding changes in mRNA levels. The molecular mechanisms driving these procancer translation programs differed on the basis of COPD severity. In mild or no COPD, the mammalian target of rapamycin pathway served as an upstream driver; whereas in advanced COPD, pathways downstream of pathological extracellular matrix emerged. Both the mammalian target of rapamycin and extracellular matrix gene expression programs paralleled activation of previously identified secretomes that can be independently induced and promote cancer initiation in mouse models. These included the senescence secretome in mild to no COPD and the ETS2 (erythroblast transformation-specific 2) secretome in moderate to severe COPD. Furthermore, in situ examination of COPD lung tissue documented stromal fibroblasts expressing key cancer-associated proteins from procancer secretomes including IL-6 (senescence secretome) in mild COPD and bone morphogenetic protein-1 (ETS2 secretome) in advanced COPD. Conclusions Two distinct stromal gene expression programs promoting cancer initiation are activated in patients with lung cancer depending on the severity of COPD as measured by lung function. Our work has implications both for screening strategies and personalized approaches to prevent cancer in COPD.

The SMARTer® Stranded Total RNA-Seq Kit v2 – Pico Input Mammalian kit from Takara® (SMARTer Pico) has proved successful and reliable in generating stranded RNA Illumina libraries from degraded total RNA and ultra-low input amounts of total RNA below detection level. Here we attempted to streamline and simplify the library prep protocol at the key fragmentation step bottleneck. Our key findings were that reduced fragmentation times neither affect the depletion efficiency, nor the library complexity. Skipping the fragmentation resulted in longer libraries when examined in the capillary electrophoresis but this was compensated for during sequencing, as long fragments are less likely to form clusters during sequencing. Skipping the fragmentation also affected the gene body coverage, where a bias to the 5’ end was observed though this compromised neither the data quality, complexity nor reproducibility. Additionally, using 16 PCR cycles seems to have little effect on the library complexity. Overall, we can see that sample input is the key to library complexity and reproducibility, while fragmentation time has less impact on data. Is total RNA fragmentation needed? Is rRNA depletion affected by fragmentation time? Or affected by input? Can we get reliable data when starting with low input?

Background Neuroblastic tumors include the neuroblastomas, ganglioneuroblastomas, and ganglioneuromas. Clinical behavior of these developmental malignancies varies from regression to aggressive growth with metastatic dissemination. Several clinical, histological, genetic, and biological features are associated with this diversity of clinical presentations. The calcium-sensing receptor (CaSR) is a G-protein coupled receptor with a key role in calcium homeostasis. We have previously reported that it is expressed in benign, differentiated neuroblastic tumors, but silenced by genetic and epigenetic events in unfavorable neuroblastomas. We have now analyzed three functionally relevant polymorphisms clustered at the signal transduction region of the CaSR (rs1801725, rs1042636 and rs1801726) to assess if genetic variants producing a less active receptor are associated with more aggressive disease course. Methods Polymorphisms were analyzed in DNA samples from 65 patients using specific Taqman Genotyping Assays. Results Mildly inactivating variant rs1801725 was associated with clinical stage 4 (P = 0.002) and the histological subgroup of undifferentiated neuroblastomas (P = 0.046). Patients harboring this polymorphism had significantly lower overall (P = 0.022) and event-free survival (P = 0.01) rates than those who were homozygous for the most common allele among Caucasians. However, this single locus genotype was not independently associated with outcome in multivariate analyses. Conversely, the tri-locus haplotype TAC was independently associated with an increased risk of death in the entire cohort (Hazard Ratio = 2.45; 95% Confidence Interval [1.14-5.29]; P = 0.022) and also in patients diagnosed with neuroblastomas (Hazard Ratio = 2.74; 95% Confidence Interval [1.20-6.25]; P = 0.016). Conclusions The TAC haplotype includes the moderately inactivating variant rs1801725 and absence of the gain-of-function rs1042636 polymorphism. Thus, its association with metastatic disease and poor outcome would add to our previous data and further support that inactivation of the CaSR gene is a mechanism associated with neuroblastoma malignant behavior.

Adaptation to cellular stresses entails an incompletely understood coordination of transcriptional and post-transcriptional gene expression programs. Here, we quantified hypoxia-dependent transcriptomes, epigenomes, and translatomes in T47D breast cancer cells and H9 human embryonic stem cells. This revealed pervasive changes in transcription start site (TSS) selection associated with nucleosome repositioning and alterations in H3K4me3 distribution. Importantly, hypoxia-associated TSS switching was induced or reversed via pharmacological modulation of H3K4me3 in the absence of hypoxia, defining a role for H3K4me3 in TSS selection independent of HIF1-transcriptional programs. By remodelling 5’UTRs, TSS switching selectively alters protein synthesis, including enhanced translation of mRNAs encoding pyruvate dehydrogenase kinase 1 (PDK1) that is essential for the metabolic adaptation to hypoxia. These results demonstrate a previously unappreciated mechanism of translational regulation during hypoxia driven by epigenetic reprogramming of the 5’UTRome.