Coordination of gene expression programs

Most cellular processes depend on the activity and interactions of proteins. The proteome, i.e. the entire set of proteins in a specific condition, is shaped by regulation of transcription, mRNA-degradation, -processing, -storage, -translation and protein degradation. Cancer cells are known to highjack gene expression processes, including the translation machinery, for their growth and survival. This occurs as a result of converging oncogenic signaling pathways which impinge on translation factors to selectively modulate synthesis of cancer-related proteins. Our understanding of mechanisms by which oncogenic pathways dynamically control their targets' translational activity is limited and could be extended by transcriptome-wide studies of changes in translation efficiency. In Paper I, we developed anota2seq which allows for statistical analysis of such data. Using a simulation approach, we showed that anota2seq constitutes an improvement compared to other methods for identification of genes under translational regulation. The relative contribution of transcriptional and translational regulation to proteome modulation has been extensively debated. This raises the interest in studies integrating data on multiple levels of gene expression regulation. In Paper II, we study the role of estrogen receptor alpha (ERα), a transcription factor that is commonly targeted in hormone-dependent cancers, in coordinating transcriptional alterations with control at the level of translation. Upon ERα depletion in a prostate cancer model, we observed massive translational offsetting whereby the translational output remains unchanged despite changes in mRNA levels. To characterize mechanisms underlying translational offsetting, we extended the scope of the anota2seq method (Paper I) to also identify genes regulated by this underappreciated mode of gene expression regulation. Next, our detailed mechanistic study revealed that upon ERα depletion, mRNAs whose levels are reduced but translationally offset have less structured 5'UTRs and are devoid of miRNA target sites and thus cannot be influenced by such translational repressors. In contrast, transcripts which were upregulated but offset at the level of translation are enriched in codons requiring U34-modified tRNAs for their translation. We finally demonstrated that ERα impacts the levels of such modified tRNAs. Cancer is a highly heterogeneous disease. In our studies of translational control, we are reaching the limits of reasonable inference when extending conclusions from experiments in cell lines into clinical settings. However, experimental methods to quantify translatomes such as polysome-profiling, are not suitable for samples with low RNA input such as tissue samples from cancer patients. Paper III presents an optimization of the polysome-profiling method, compares it with the classical approach and validates that this new approach is suitable to study novel mechanisms regulating mRNA translation in large collections of tissue samples

The Protozoan Parasite Toxoplasma gondii Selectively Reprograms the Host Cell Translatome

The intracellular parasite Toxoplasma gondii promotes infection by targeting multiple host cell processes; however, whether it modulates mRNA translation is currently unknown. Here, we show that infection of primary murine macrophages with type I or II T. gondii strains causes a profound perturbation of the host cell translatome. Notably, translation of transcripts encoding proteins involved in metabolic activity and components of the translation machinery was activated upon infection. In contrast, the translational efficiency of mRNAs related to immune cell activation and cytoskeleton/cytoplasm organization was largely suppressed. Mechanistically, T. gondii bolstered mechanistic target of rapamycin (mTOR) signaling to selectively activate the translation of mTOR-sensitive mRNAs, including those with a 5'-terminal oligopyrimidine (5' TOP) motif and those encoding mitochondrion-related proteins. Consistent with parasite modulation of host mTOR-sensitive translation to promote infection, inhibition of mTOR activity suppressed T. gondii replication. Thus, selective reprogramming of host mRNA translation represents an important subversion strategy during T. gondii infection

Exploratory Analysis of Multiple Omics Datasets Using the Adjusted RV Coefficient

The integration of multiple high-dimensional data sets (omics data) has been a very active but challenging area of bioinformatics research in recent years. Various adaptations of non-standard multivariate statistical tools have been suggested that allow to analyze and visualize such data sets simultaneously. However, these methods typically can deal with two data sets only, whereas systems biology experiments often generate larger numbers of high-dimensional data sets. For this reason, we suggest an explorative analysis of similarity between data sets as an initial analysis steps. This analysis is based on the RV coefficient, a matrix correlation, that can be interpreted as a generalization of the squared correlation from two single variables to two sets of variables. It has been shown before however that the high-dimensionality of the data introduces substantial bias to the RV.We therefore introduce an alternative version, the adjusted RV, which is unbiased in the case of independent data sets. We can also show that in many situations, particularly for very high-dimensional data sets, the adjusted RV is a better estimator than previously RV versions in terms of the mean square error and the power of the independence test based on it.

Generally applicable transcriptome-wide analysis of translation using anota2seq

mRNA translation plays an evolutionarily conserved role in homeostasis and when dysregulated contributes to various disorders including metabolic and neurological diseases and cancer. Notwithstanding that optimal and universally applicable methods are critical for understanding the complex role of translational control under physiological and pathological conditions, approaches to analyze translatomes are largely underdeveloped. To address this, we developed the anota2seq algorithm which outperforms current methods for statistical identification of changes in translation. Notably, in contrast to available analytical methods, anota2seq also allows specific identification of an underappreciated mode of gene expression regulation whereby translation acts as a buffering mechanism which maintains protein levels despite fluctuations in corresponding mRNA abundance ('translational buffering'). Thus, the universal anota2seq algorithm allows efficient and hitherto unprecedented interrogation of translatomes which is anticipated to advance knowledge regarding the role of translation in homeostasis and disease

IL-15 activates mTOR and primes stress-activated gene expression leading to prolonged antitumor capacity of NK cells.

Treatment of hematological malignancies by adoptive transfer of activated natural killer (NK) cells is limited by poor postinfusion persistence. We compared the ability of interleukin-2 (IL-2) and IL-15 to sustain human NK-cell functions following cytokine withdrawal to model postinfusion performance. In contrast to IL-2, IL-15 mediated stronger signaling through the IL-2/15 receptor complex and provided cell function advantages. Genome-wide analysis of cytosolic and polysome-associated messenger RNA (mRNA) revealed not only cytokine-dependent differential mRNA levels and translation during cytokine activation but also that most gene expression differences were primed by IL-15 and only manifested after cytokine withdrawal. IL-15 augmented mammalian target of rapamycin (mTOR) signaling, which correlated with increased expression of genes related to cell metabolism and respiration. Consistently, mTOR inhibition abrogated IL-15-induced cell function advantages. Moreover, mTOR-independent STAT-5 signaling contributed to improved NK-cell function during cytokine activation but not following cytokine withdrawal. The superior performance of IL-15-stimulated NK cells was also observed using a clinically applicable protocol for NK-cell expansion in vitro and in vivo. Finally, expression of IL-15 correlated with cytolytic immune functions in patients with B-cell lymphoma and favorable clinical outcome. These findings highlight the importance of mTOR-regulated metabolic processes for immune cell functions and argue for implementation of IL-15 in adoptive NK-cell cancer therapy

Analysis of human lung mast cells by single cell RNA sequencing

Mast cells are tissue-resident cells playing major roles in homeostasis and disease conditions. Lung mast cells are particularly important in airway inflammatory diseases such as asthma. Human mast cells are classically divided into the subsets MCT and MCTC, where MCT express the mast cell protease tryptase and MCTC in addition express chymase, carboxypeptidase A3 (CPA3) and cathepsin G. Apart from the disctintion of the MCT and MCTC subsets, little is known about the heterogeniety of human lung mast cells and a deep analysis of their heterogeniety has previously not been performed. We therefore performed single cell RNA sequencing on sorted human lung mast cells using SmartSeq2. The mast cells showed high expression of classical mast cell markers. The expression of several individual genes varied considerably among the cells, however, no subpopulations were detected by unbiased clustering. Variable genes included the protease-encoding transcripts CMA1 (chymase) and CTSG (cathepsin G). Human lung mast cells are predominantly of the MCT subset and consistent with this, the expression of CMA1 was only detectable in a small proportion of the cells, and correlated moderately to CTSG. However, in contrast to established data for the protein, CPA3 mRNA was high in all cells and the correlation of CPA3 to CMA1 was weak

eIF4E-Binding Proteins 1 and 2 Limit Macrophage Anti-Inflammatory Responses through Translational Repression of IL-10 and Cyclooxygenase-2

Macrophages represent one of the first lines of defense during infections and are essential for resolution of inflammation following pathogen clearance. Rapid activation or suppression of protein synthesis via changes in translational efficiency allows cells of the immune system, including macrophages, to quickly respond to external triggers or cues without de novo mRNA synthesis. The translational repressors eIF4E-binding proteins 4E-BP1 and 4E-BP2 (4E-BP1/2) are central regulators of proinflammatory cytokine synthesis during viral and parasitic infections. However, it remains to be established whether 4E-BP1/2 play a role in translational control of anti-inflammatory responses. By comparing translational efficiencies of immune-related transcripts in macrophages from wild-type and 4E-BP1/2 double-knockout mice, we found that translation of mRNAs encoding two major regulators of inflammation, IL-10 and PG-endoperoxide synthase 2/cyclooxygenase-2, is controlled by 4E-BP1/2. Genetic deletion of 4E-BP1/2 in macrophages increased endogenous IL-10 and PGE2 protein synthesis in response to TLR4 stimulation and reduced their bactericidal capacity. The molecular mechanism involves enhanced anti-inflammatory gene expression (sIl1ra, Nfil3, Arg1, Serpinb2) owing to upregulation of IL-10-STAT3 and PGE(2)-C/EBP beta signaling. These data provide evidence that 4E-BP1/2 limit anti-inflammatory responses in macrophages and suggest that dysregulated activity of 4E-BP1/2 might be involved in reprogramming of the translational and downstream transcriptional landscape of macrophages during pathological conditions, such as infections and cancer

Randomized phase II/III trial assessing gemcitabine/carboplatin and methotrexate/carboplatin/vinblastine in patients with advanced urothelial cancer who are unfit for cisplatin-based chemotherapy: EORTC study 30986.

This is the first randomized phase II/III trial comparing two carboplatin-based chemotherapy regimens in patients with urothelial cancer who are ineligible ("unfit") for cisplatin chemotherapy.Clinical Trial, Phase IIClinical Trial, Phase IIIJournal ArticleMulticenter StudyRandomized Controlled TrialResearch Support, N.I.H. ExtramuralResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe