E2F transcription factor-1 modulates expression of glutamine metabolic genes in mouse embryonic fibroblasts and uterine sarcoma cells.

Metabolic reprogramming is considered as a hallmark of cancer and is clinically exploited as a novel target for therapy. The E2F transcription factor-1 (E2F1) regulates various cellular processes, including proliferative and metabolic pathways, and acts, depending on the cellular and molecular context, as an oncogene or tumor suppressor. The latter is evident by the observation that E2f1-knockout mice develop spontaneous tumors, including uterine sarcomas. This dual role warrants a detailed investigation of how E2F1 loss impacts metabolic pathways related to cancer progression. Our data indicate that E2F1 binds to the promoter of several glutamine metabolism-related genes. Interestingly, the expression of genes in the glutamine metabolic pathway were increased in mouse embryonic fibroblasts (MEFs) lacking E2F1. In addition, we confirm that E2f1-/- MEFs are more efficient in metabolizing glutamine and producing glutamine-derived precursors for proliferation. Mechanistically, we observe a co-occupancy of E2F1 and MYC on glutamine metabolic promoters, increased MYC binding after E2F1 depletion and that silencing of MYC decreased the expression of glutamine-related genes in E2f1-/- MEFs. Analyses of transcriptomic profiles in 29 different human cancers identified uterine sarcoma that showed a negative correlation between E2F1 and glutamine metabolic genes. CRISPR/Cas9 knockout of E2F1 in the uterine sarcoma cell line SK-UT-1 confirmed elevated glutamine metabolic gene expression, increased proliferation and increased MYC binding to glutamine-related promoters upon E2F1 loss. Together, our data suggest a crucial role of E2F1 in energy metabolism and metabolic adaptation in uterine sarcoma cells.

Comprehensive map of ribosomal 2'-O-methylation and C/D box snoRNAs in Drosophila melanogaster.

During their maturation, ribosomal RNAs (rRNAs) are decorated by hundreds of chemical modifications that participate in proper folding of rRNA secondary structures and therefore in ribosomal function. Along with pseudouridine, methylation of the 2'-hydroxyl ribose moiety (Nm) is the most abundant modification of rRNAs. The majority of Nm modifications in eukaryotes are placed by Fibrillarin, a conserved methyltransferase belonging to a ribonucleoprotein complex guided by C/D box small nucleolar RNAs (C/D box snoRNAs). These modifications impact interactions between rRNAs, tRNAs and mRNAs, and some are known to fine tune translation rates and efficiency. In this study, we built the first comprehensive map of Nm sites in Drosophila melanogaster rRNAs using two complementary approaches (RiboMethSeq and Nanopore direct RNA sequencing) and identified their corresponding C/D box snoRNAs by whole-transcriptome sequencing. We de novo identified 61 Nm sites, from which 55 are supported by both sequencing methods, we validated the expression of 106 C/D box snoRNAs and we predicted new or alternative rRNA Nm targets for 31 of them. Comparison of methylation level upon different stresses show only slight but specific variations, indicating that this modification is relatively stable in D. melanogaster. This study paves the way to investigate the impact of snoRNA-mediated 2'-O-methylation on translation and proteostasis in a whole organism.

ROS-induced ribosome impairment underlies ZAKα-mediated metabolic decline in obesity and aging.

The ribotoxic stress response (RSR) is a signaling pathway in which the p38- and c-Jun N-terminal kinase (JNK)-activating mitogen-activated protein kinase kinase kinase (MAP3K) ZAKα senses stalling and/or collision of ribosomes. Here, we show that reactive oxygen species (ROS)-generating agents trigger ribosomal impairment and ZAKα activation. Conversely, zebrafish larvae deficient for ZAKα are protected from ROS-induced pathology. Livers of mice fed a ROS-generating diet exhibit ZAKα-activating changes in ribosomal elongation dynamics. Highlighting a role for the RSR in metabolic regulation, ZAK-knockout mice are protected from developing high-fat high-sugar (HFHS) diet-induced blood glucose intolerance and liver steatosis. Finally, ZAK ablation slows animals from developing the hallmarks of metabolic aging. Our work highlights ROS-induced ribosomal impairment as a physiological activation signal for ZAKα that underlies metabolic adaptation in obesity and aging.

Molecular basis of translation termination at noncanonical stop codons in human mitochondria.

The genetic code that specifies the identity of amino acids incorporated into proteins during protein synthesis is almost universally conserved. Mitochondrial genomes feature deviations from the standard genetic code, including the reassignment of two arginine codons to stop codons. The protein required for translation termination at these noncanonical stop codons to release the newly synthesized polypeptides is not currently known. In this study, we used gene editing and ribosomal profiling in combination with cryo-electron microscopy to establish that mitochondrial release factor 1 (mtRF1) detects noncanonical stop codons in human mitochondria by a previously unknown mechanism of codon recognition. We discovered that binding of mtRF1 to the decoding center of the ribosome stabilizes a highly unusual conformation in the messenger RNA in which the ribosomal RNA participates in specific recognition of the noncanonical stop codons.

A conditional Smg6 mutant mouse model reveals circadian clock regulation through the nonsense-mediated mRNA decay pathway.

Nonsense-mediated messenger RNA (mRNA) decay (NMD) has been intensively studied as a surveillance pathway that degrades erroneous transcripts arising from mutations or RNA processing errors. While additional roles in physiological control of mRNA stability have emerged, possible functions in mammalian physiology in vivo remain unclear. Here, we created a conditional mouse allele that allows converting the NMD effector nuclease SMG6 from wild-type to nuclease domain-mutant protein. We find that NMD down-regulation affects the function of the circadian clock, a system known to require rapid mRNA turnover. Specifically, we uncover strong lengthening of free-running circadian periods for liver and fibroblast clocks and direct NMD regulation of Cry2 mRNA, encoding a key transcriptional repressor within the rhythm-generating feedback loop. Transcriptome-wide changes in daily mRNA accumulation patterns in the entrained liver, as well as an altered response to food entrainment, expand the known scope of NMD regulation in mammalian gene expression and physiology.

Ribosome stalling is a signal for metabolic regulation by the ribotoxic stress response.

Impairment of translation can lead to collisions of ribosomes, which constitute an activation platform for several ribosomal stress-surveillance pathways. Among these is the ribotoxic stress response (RSR), where ribosomal sensing by the MAP3K ZAKα leads to activation of p38 and JNK kinases. Despite these insights, the physiological ramifications of ribosomal impairment and downstream RSR signaling remain elusive. Here, we show that stalling of ribosomes is sufficient to activate ZAKα. In response to amino acid deprivation and full nutrient starvation, RSR impacts on the ensuing metabolic responses in cells, nematodes, and mice. The RSR-regulated responses in these model systems include regulation of AMPK and mTOR signaling, survival under starvation conditions, stress hormone production, and regulation of blood sugar control. In addition, ZAK-/- male mice present a lean phenotype. Our work highlights impaired ribosomes as metabolic signals and demonstrates a role for RSR signaling in metabolic regulation.

Analysis of Eukaryotic lincRNA Sequences Indicates Signatures of Hindered Translation Linked to Selection Pressure.

Long intergenic noncoding RNAs (lincRNAs) represent a large fraction of transcribed loci in eukaryotic genomes. Although classified as noncoding, most lincRNAs contain open reading frames (ORFs), and it remains unclear why cytoplasmic lincRNAs are not or very inefficiently translated. Here, we analyzed signatures of hindered translation in lincRNA sequences from five eukaryotes, covering a range of natural selection pressures. In fission yeast and Caenorhabditis elegans, that is, species under strong selection, we detected significantly shorter ORFs, a suboptimal sequence context around start codons for translation initiation, and trinucleotides ("codons") corresponding to less abundant tRNAs than for neutrally evolving control sequences, likely impeding translation elongation. For human, we detected signatures for cell-type-specific hindrance of lincRNA translation, in particular codons in abundant cytoplasmic lincRNAs corresponding to lower expressed tRNAs than control codons, in three out of five human cell lines. We verified that varying tRNA expression levels between cell lines are reflected in the amount of ribosomes bound to cytoplasmic lincRNAs in each cell line. We further propose that codons at ORF starts are particularly important for reducing ribosome-binding to cytoplasmic lincRNA ORFs. Altogether, our analyses indicate that in species under stronger selection lincRNAs evolved sequence features generally hindering translation and support cell-type-specific hindrance of translation efficiency in human lincRNAs. The sequence signatures we have identified may improve predicting peptide-coding and genuine noncoding lincRNAs in a cell type.

Computational identification and experimental characterization of preferred downstream positions in human core promoters.

Metazoan core promoters, which direct the initiation of transcription by RNA polymerase II (Pol II), may contain short sequence motifs termed core promoter elements/motifs (e.g. the TATA box, initiator (Inr) and downstream core promoter element (DPE)), which recruit Pol II via the general transcription machinery. The DPE was discovered and extensively characterized in Drosophila, where it is strictly dependent on both the presence of an Inr and the precise spacing from it. Since the Drosophila DPE is recognized by the human transcription machinery, it is most likely that some human promoters contain a downstream element that is similar, though not necessarily identical, to the Drosophila DPE. However, only a couple of human promoters were shown to contain a functional DPE, and attempts to computationally detect human DPE-containing promoters have mostly been unsuccessful. Using a newly-designed motif discovery strategy based on Expectation-Maximization probabilistic partitioning algorithms, we discovered preferred downstream positions (PDP) in human promoters that resemble the Drosophila DPE. Available chromatin accessibility footprints revealed that Drosophila and human Inr+DPE promoter classes are not only highly structured, but also similar to each other, particularly in the proximal downstream region. Clustering of the corresponding sequence motifs using a neighbor-joining algorithm strongly suggests that canonical Inr+DPE promoters could be common to metazoan species. Using reporter assays we demonstrate the contribution of the identified downstream positions to the function of multiple human promoters. Furthermore, we show that alteration of the spacing between the Inr and PDP by two nucleotides results in reduced promoter activity, suggesting a spacing dependency of the newly discovered human PDP on the Inr. Taken together, our strategy identified novel functional downstream positions within human core promoters, supporting the existence of DPE-like motifs in human promoters.

Structural basis of ribosomal frameshifting during translation of the SARS-CoV-2 RNA genome.

Programmed ribosomal frameshifting is a key event during translation of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA genome that allows synthesis of the viral RNA-dependent RNA polymerase and downstream proteins. Here, we present the cryo-electron microscopy structure of a translating mammalian ribosome primed for frameshifting on the viral RNA. The viral RNA adopts a pseudoknot structure that lodges at the entry to the ribosomal messenger RNA (mRNA) channel to generate tension in the mRNA and promote frameshifting, whereas the nascent viral polyprotein forms distinct interactions with the ribosomal tunnel. Biochemical experiments validate the structural observations and reveal mechanistic and regulatory features that influence frameshifting efficiency. Finally, we compare compounds previously shown to reduce frameshifting with respect to their ability to inhibit SARS-CoV-2 replication, establishing coronavirus frameshifting as a target for antiviral intervention.

Low expression of the PPARγ-regulated gene thioredoxin-interacting protein accompanies human melanoma progression and promotes experimental lung metastases.

The thioredoxin system plays key roles in regulating cancer cell malignancy. Here we identify the Thioredoxin-interacting protein (TXNIP) as a gene, which expression is regulated by PPARγ in melanoma cells. We show that high TXNIP expression levels associate with benign melanocytic lesions, with tumor regression in patients on MAP kinase targeted therapy, with decreased proliferation in patients' melanoma biopsies, and with cell cycle arrest in human melanoma cell lines. In contrast, reduced TXNIP expression associates with advanced melanoma and with disease progression in patients. TXNIP depletion in human melanoma cells altered the expression of integrin beta-3 and the localization of the integrin alpha-v/beta-3 dimer at their surface. Moreover, TXNIP depletion affected human melanoma cell motility and improved their capacity to colonize mouse lungs in an in vivo assay. This study establishes TXNIP as a PPARγ-regulated gene in melanoma cells, thereby suggesting a link between these two proteins both involved in the regulation of cancer and of energy metabolism. It also reveals that the decrease in TXNIP expression, which is observed in advanced patient tumors, likely favors lung metastatic seeding of malignant cells.

Jasmonate biosynthesis arising from altered cell walls is prompted by turgor-driven mechanical compression.

Despite the vital roles of jasmonoyl-isoleucine (JA-Ile) in governing plant growth and environmental acclimation, it remains unclear what intracellular processes lead to its induction. Here, we provide compelling genetic evidence that mechanical and osmotic regulation of turgor pressure represents a key elicitor of JA-Ile biosynthesis. After identifying cell wall mutant alleles in KORRIGAN1 (KOR1) with elevated JA-Ile in seedling roots, we found that ectopic JA-Ile resulted from cell nonautonomous signals deriving from enlarged cortex cells compressing inner tissues and stimulating JA-Ile production. Restoring cortex cell size by cell type-specific KOR1 complementation, by isolating a genetic kor1 suppressor, and by lowering turgor pressure with hyperosmotic treatments abolished JA-Ile signaling. Conversely, hypoosmotic treatment activated JA-Ile signaling in wild-type plants. Furthermore, constitutive JA-Ile levels guided mutant roots toward greater water availability. Collectively, these findings enhance our understanding on JA-Ile biosynthesis initiation and reveal a previously undescribed role of JA-Ile in orchestrating environmental resilience.

Translation is required for miRNA-dependent decay of endogenous transcripts.

Post-transcriptional repression of gene expression by miRNAs occurs through transcript destabilization or translation inhibition. mRNA decay is known to account for most miRNA-dependent repression. However, because transcript decay occurs co-translationally, whether target translation is a requirement for miRNA-dependent transcript destabilization remains unknown. To decouple these two molecular processes, we used cytosolic long noncoding RNAs (lncRNAs) as models for endogenous transcripts that are not translated. We show that, despite interacting with the miRNA-loaded RNA-induced silencing complex, the steady-state abundance and decay rates of these transcripts are minimally affected by miRNA loss. To further validate the apparent requirement of translation for miRNA-dependent decay, we fused two lncRNA candidates to the 3'-end of a protein-coding gene reporter and found this results in their miRNA-dependent destabilization. Further analysis revealed that the few natural lncRNAs whose levels are regulated by miRNAs in mESCs tend to associate with translating ribosomes, and possibly represent misannotated micropeptides, further substantiating the necessity of target translation for miRNA-dependent transcript decay. In summary, our analyses suggest that translation is required for miRNA-dependent transcript destabilization, and demonstrate that the levels of coding and noncoding transcripts are differently affected by miRNAs.

Readthrough of stop codons under limiting ABCE1 concentration involves frameshifting and inhibits nonsense-mediated mRNA decay.

To gain insight into the mechanistic link between translation termination and nonsense-mediated mRNA decay (NMD), we depleted the ribosome recycling factor ABCE1 in human cells, resulting in an upregulation of NMD-sensitive mRNAs. Suppression of NMD on these mRNAs occurs prior to their SMG6-mediated endonucleolytic cleavage. ABCE1 depletion caused ribosome stalling at termination codons (TCs) and increased ribosome occupancy in 3' UTRs, implying enhanced TC readthrough. ABCE1 knockdown indeed increased the rate of readthrough and continuation of translation in different reading frames, providing a possible explanation for the observed NMD inhibition, since enhanced readthrough displaces NMD activating proteins from the 3' UTR. Our results indicate that stalling at TCs triggers ribosome collisions and activates ribosome quality control. Collectively, we show that improper translation termination can lead to readthrough of the TC, presumably due to ribosome collisions pushing the stalled ribosomes into the 3' UTR, where it can resume translation in-frame as well as out-of-frame.

Human NMD ensues independently of stable ribosome stalling.

Nonsense-mediated mRNA decay (NMD) is a translation-dependent RNA degradation pathway that is important for the elimination of faulty, and the regulation of normal, mRNAs. The molecular details of the early steps in NMD are not fully understood but previous work suggests that NMD activation occurs as a consequence of ribosome stalling at the termination codon (TC). To test this hypothesis, we established an in vitro translation-coupled toeprinting assay based on lysates from human cells that allows monitoring of ribosome occupancy at the TC of reporter mRNAs. In contrast to the prevailing NMD model, our in vitro system reveals similar ribosomal occupancy at the stop codons of NMD-sensitive and NMD-insensitive reporter mRNAs. Moreover, ribosome profiling reveals a similar density of ribosomes at the TC of endogenous NMD-sensitive and NMD-insensitive mRNAs in vivo. Together, these data show that NMD activation is not accompanied by stable stalling of ribosomes at TCs.

Transcriptome-wide sites of collided ribosomes reveal principles of translational pausing.

Translation initiation is the major regulatory step defining the rate of protein production from an mRNA. Meanwhile, the impact of nonuniform ribosomal elongation rates is largely unknown. Using a modified ribosome profiling protocol based on footprints from two closely packed ribosomes (disomes), we have mapped ribosomal collisions transcriptome-wide in mouse liver. We uncover that the stacking of an elongating onto a paused ribosome occurs frequently and scales with translation rate, trapping ∼10% of translating ribosomes in the disome state. A distinct class of pause sites is indicative of deterministic pausing signals. Pause site association with specific amino acids, peptide motifs, and nascent polypeptide structure is suggestive of programmed pausing as a widespread mechanism associated with protein folding. Evolutionary conservation at disome sites indicates functional relevance of translational pausing. Collectively, our disome profiling approach allows unique insights into gene regulation occurring at the step of translation elongation.

CDK7 Mediates the Beta-Adrenergic Signaling in Thermogenic Brown and White Adipose Tissues.

Cyclin-dependent kinases (CDKs) are emerging regulators of adipose tissue metabolism. Here we aimed to explore the role of CDK7 in thermogenic fat. We found that CDK7 brown adipose tissue (BAT)-specific knockout mice (Cdk7bKO) have decreased BAT mass and impaired ß3-adrenergic signaling and develop hypothermia upon cold exposure. We found that loss of CDK7 in BAT disrupts the induction of thermogenic genes in response to cold. However, Cdk7bKO mice do not show systemic metabolic dysfunction. Increased expression of genes of the creatine metabolism compensates for the heat generation in the BAT of Cdk7bKO mice in response to cold. Finally, we show that CDK7 is required for beta 3-adrenergic agonist-induced browning of white adipose tissue (WAT). Indeed, Cdk7 ablation in all adipose tissues (Cdk7aKO) has impaired browning in WAT. Together, our results demonstrate that CDK7 is an important mediator of beta-adrenergic signaling in thermogenic brown and beige fat.

A Reservoir of Pluripotent Phloem Cells Safeguards the Linear Developmental Trajectory of Protophloem Sieve Elements.

Plant cells can change their identity based on positional information, a mechanism that confers developmental plasticity to plants. This ability, common to distinct multicellular organisms, is particularly relevant for plant phloem cells. Protophloem sieve elements (PSEs), one type of phloem conductive cells, act as the main organizers of the phloem pole, which comprises four distinct cell files organized in a conserved pattern. Here, we report how Arabidopsis roots generate a reservoir of meristematic phloem cells competent to swap their cell identities. Although PSE misspecification induces cell identity hybridism, the activity of RECEPTOR LIKE PROTEIN KINASE 2 (RPK2) by perceiving CLE45 peptide contributes to restrict PSE identity to the PSE position. By maintaining a spatiotemporal window when PSE and PSE-adjacent cells' identities are interchangeable, CLE45 signaling endows phloem cells with the competence to re-pattern a functional phloem pole when protophloem fails to form.

EPD in 2020: enhanced data visualization and extension to ncRNA promoters.

The Eukaryotic Promoter Database (EPD), available online at https://epd.epfl.ch, provides accurate transcription start site (TSS) information for promoters of 15 model organisms plus corresponding functional genomics data that can be viewed in a genome browser, queried or analyzed via web interfaces, or exported in standard formats (FASTA, BED, CSV) for subsequent analysis with other tools. Recent work has focused on the improvement of the EPD promoter viewers, which use the UCSC Genome Browser as visualization platform. Thousands of high-resolution tracks for CAGE, ChIP-seq and similar data have been generated and organized into public track hubs. Customized, reproducible promoter views, combining EPD-supplied tracks with native UCSC Genome Browser tracks, can be accessed from the organism summary pages or from individual promoter entries. Moreover, thanks to recent improvements and stabilization of ncRNA gene catalogs, we were able to release promoter collections for certain classes of ncRNAs from human and mouse. Furthermore, we developed automatic computational protocols to assign orphan TSS peaks to downstream genes based on paired-end (RAMPAGE) TSS mapping data, which enabled us to add nearly 9000 new entries to the human promoter collection. Since our last article in this journal, EPD was extended to five more model organisms: rhesus monkey, rat, dog, chicken and Plasmodium falciparum.

Opposing chromatin remodelers control transcription initiation frequency and start site selection.

Precise nucleosome organization at eukaryotic promoters is thought to be generated by multiple chromatin remodeler (CR) enzymes and to affect transcription initiation. Using an integrated analysis of chromatin remodeler binding and nucleosome occupancy following rapid remodeler depletion, we investigated the interplay between these enzymes and their impact on transcription in yeast. We show that many promoters are affected by multiple CRs that operate in concert or in opposition to position the key transcription start site (TSS)-associated +1 nucleosome. We also show that nucleosome movement after CR inactivation usually results from the activity of another CR and that in the absence of any remodeling activity, +1 nucleosomes largely maintain their positions. Finally, we present functional assays suggesting that +1 nucleosome positioning often reflects a trade-off between maximizing RNA polymerase recruitment and minimizing transcription initiation at incorrect sites. Our results provide a detailed picture of fundamental mechanisms linking promoter nucleosome architecture to transcription initiation.

MGA repository: a curated data resource for ChIP-seq and other genome annotated data.

The Mass Genome Annotation (MGA) repository is a resource designed to store published next generation sequencing data and other genome annotation data (such as gene start sites, SNPs, etc.) in a completely standardised format. Each sample has undergone local processing in order the meet the strict MGA format requirements. The original data source, the reformatting procedure and the biological characteristics of the samples are described in an accompanying documentation file manually edited by data curators. 10 model organisms are currently represented: Homo sapiens, Mus musculus, Danio rerio, Drosophila melanogaster, Apis mellifera, Caenorhabditis elegans, Arabidopsis thaliana, Zea mays, Saccharomyces cerevisiae and Schizosaccharomyces pombe. As of today, the resource contains over 24 000 samples. In conjunction with other tools developed by our group (the ChIP-Seq and SSA servers), it allows users to carry out a great variety of analysis task with MGA samples, such as making aggregation plots and heat maps for selected genomic regions, finding peak regions, generating custom tracks for visualizing genomic features in a UCSC genome browser window, or downloading chromatin data in a table format suitable for local processing with more advanced statistical analysis software such as R. Home page: http://ccg.vital-it.ch/mga/.