Diurnal Oscillations in Liver Mass and Cell Size Accompany Ribosome Assembly Cycles.

The liver plays a pivotal role in metabolism and xenobiotic detoxification, processes that must be particularly efficient when animals are active and feed. A major question is how the liver adapts to these diurnal changes in physiology. Here, we show that, in mice, liver mass, hepatocyte size, and protein levels follow a daily rhythm, whose amplitude depends on both feeding-fasting and light-dark cycles. Correlative evidence suggests that the daily oscillation in global protein accumulation depends on a similar fluctuation in ribosome number. Whereas rRNA genes are transcribed at similar rates throughout the day, some newly synthesized rRNAs are polyadenylated and degraded in the nucleus in a robustly diurnal fashion with a phase opposite to that of ribosomal protein synthesis. Based on studies with cultured fibroblasts, we propose that rRNAs not packaged into complete ribosomal subunits are polyadenylated by the poly(A) polymerase PAPD5 and degraded by the nuclear exosome.

Mammalian RNA Decay Pathways Are Highly Specialized and Widely Linked to Translation.

RNA decay is crucial for mRNA turnover and surveillance and misregulated in many diseases. This complex system is challenging to study, particularly in mammals, where it remains unclear whether decay pathways perform specialized versus redundant roles. Cytoplasmic pathways and links to translation are particularly enigmatic. By directly profiling decay factor targets and normal versus aberrant translation in mouse embryonic stem cells (mESCs), we uncovered extensive decay pathway specialization and crosstalk with translation. XRN1 (5'-3') mediates cytoplasmic bulk mRNA turnover whereas SKIV2L (3'-5') is universally recruited by ribosomes, tackling aberrant translation and sometimes modulating mRNA abundance. Further exploring translation surveillance revealed AVEN and FOCAD as SKIV2L interactors. AVEN prevents ribosome stalls at structured regions, which otherwise require SKIV2L for clearance. This pathway is crucial for histone translation, upstream open reading frame (uORF) regulation, and counteracting ribosome arrest on small ORFs. In summary, we uncovered key targets, components, and functions of mammalian RNA decay pathways and extensive coupling to translation.

Transcriptome and translatome co-evolution in mammals.

Gene-expression programs define shared and species-specific phenotypes, but their evolution remains largely uncharacterized beyond the transcriptome layer1. Here we report an analysis of the co-evolution of translatomes and transcriptomes using ribosome-profiling and matched RNA-sequencing data for three organs (brain, liver and testis) in five mammals (human, macaque, mouse, opossum and platypus) and a bird (chicken). Our within-species analyses reveal that translational regulation is widespread in the different organs, in particular across the spermatogenic cell types of the testis. The between-species divergence in gene expression is around 20% lower at the translatome layer than at the transcriptome layer owing to extensive buffering between the expression layers, which especially preserved old, essential and housekeeping genes. Translational upregulation specifically counterbalanced global dosage reductions during the evolution of sex chromosomes and the effects of meiotic sex-chromosome inactivation during spermatogenesis. Despite the overall prevalence of buffering, some genes evolved faster at the translatome layer-potentially indicating adaptive changes in expression; testis tissue shows the highest fraction of such genes. Further analyses incorporating mass spectrometry proteomics data establish that the co-evolution of transcriptomes and translatomes is reflected at the proteome layer. Together, our work uncovers co-evolutionary patterns and associated selective forces across the expression layers, and provides a resource for understanding their interplay in mammalian organs.

Global and precise identification of functional miRNA targets in mESCs by integrative analysis.

MicroRNA (miRNA) loaded Argonaute (AGO) complexes regulate gene expression via direct base pairing with their mRNA targets. Previous works suggest that up to 60% of mammalian transcripts might be subject to miRNA-mediated regulation, but it remains largely unknown which fraction of these interactions are functional in a specific cellular context. Here, we integrate transcriptome data from a set of miRNA-depleted mouse embryonic stem cell (mESC) lines with published miRNA interaction predictions and AGO-binding profiles. Using this integrative approach, combined with molecular validation data, we present evidence that < 10% of expressed genes are functionally and directly regulated by miRNAs in mESCs. In addition, analyses of the stem cell-specific miR-290-295 cluster target genes identify TFAP4 as an important transcription factor for early development. The extensive datasets developed in this study will support the development of improved predictive models for miRNA-mRNA functional interactions.

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.

SIRT1 regulates circadian clock gene expression through PER2 deacetylation.

The mammalian circadian timing system is composed of a central pacemaker in the suprachiasmatic nucleus of the brain that synchronizes countless subsidiary oscillators in peripheral tissues. The rhythm-generating mechanism is thought to rely on a feedback loop involving positively and negatively acting transcription factors. BMAL1 and CLOCK activate the expression of Period (Per) and Cryptochrome (Cry) genes, and once PER and CRY proteins accumulate to a critical level they form complexes with BMAL1-CLOCK heterodimers and thereby repress the transcription of their own genes. Here, we show that SIRT1, an NAD(+)-dependent protein deacetylase, is required for high-magnitude circadian transcription of several core clock genes, including Bmal1, Rorgamma, Per2, and Cry1. SIRT1 binds CLOCK-BMAL1 in a circadian manner and promotes the deacetylation and degradation of PER2. Given the NAD(+) dependence of SIRT1 deacetylase activity, it is likely that SIRT1 connects cellular metabolism to the circadian core clockwork circuitry.

Charting DENR-dependent translation reinitiation uncovers predictive uORF features and links to circadian timekeeping via Clock.

The non-canonical initiation factor DENR promotes translation reinitiation on mRNAs harbouring upstream open reading frames (uORFs). Moreover, DENR depletion shortens circadian period in mouse fibroblasts, suggesting involvement of uORF usage and reinitiation in clock regulation. To identify DENR-regulated translation events transcriptome-wide and, in particular, specific core clock transcripts affected by this mechanism, we have used ribosome profiling in DENR-deficient NIH3T3 cells. We uncovered 240 transcripts with altered translation rate, and used linear regression analysis to extract 5' UTR features predictive of DENR dependence. Among core clock genes, we identified Clock as a DENR target. Using Clock 5' UTR mutants, we mapped the specific uORF through which DENR acts to regulate CLOCK protein biosynthesis. Notably, these experiments revealed an alternative downstream start codon, likely representing the bona fide CLOCK N-terminus. Our findings provide insights into uORF-mediated translational regulation that can regulate the mammalian circadian clock and gene expression at large.

Design of in vitro Transcribed mRNA Vectors for Research and Therapy.

The use of in vitro transcribed messenger RNA (ivt mRNA) for vaccination, gene therapy and cell reprograming has become increasingly popular in research and medicine. This method can be used in vitro (transfected in cells) or administered naked or formulated (lipoplexes, polyplexes, and lipopolyplexes that deliver the RNA to specific organs, such as immune structures, the lung or liver) and is designed to be an immunostimulatory or immunosilent agent. This vector contains several functional regions (Cap, 5' untranslated region, open reading frame, 3' untranslated region and poly-A tail) that can all be optimised to generate a highly efficacious ivt mRNA. In this study, we review these aspects and report on the effect of the ivt mRNA purification method on the functionality of this synthetic transient genetic vector.

Ribosome profiling reveals the rhythmic liver translatome and circadian clock regulation by upstream open reading frames.

Mammalian gene expression displays widespread circadian oscillations. Rhythmic transcription underlies the core clock mechanism, but it cannot explain numerous observations made at the level of protein rhythmicity. We have used ribosome profiling in mouse liver to measure the translation of mRNAs into protein around the clock and at high temporal and nucleotide resolution. We discovered, transcriptome-wide, extensive rhythms in ribosome occupancy and identified a core set of approximately 150 mRNAs subject to particularly robust daily changes in translation efficiency. Cycling proteins produced from nonoscillating transcripts revealed thus-far-unknown rhythmic regulation associated with specific pathways (notably in iron metabolism, through the rhythmic translation of transcripts containing iron responsive elements), and indicated feedback to the rhythmic transcriptome through novel rhythmic transcription factors. Moreover, estimates of relative levels of core clock protein biosynthesis that we deduced from the data explained known features of the circadian clock better than did mRNA expression alone. Finally, we identified uORF translation as a novel regulatory mechanism within the clock circuitry. Consistent with the occurrence of translated uORFs in several core clock transcripts, loss-of-function of Denr, a known regulator of reinitiation after uORF usage and of ribosome recycling, led to circadian period shortening in cells. In summary, our data offer a framework for understanding the dynamics of translational regulation, circadian gene expression, and metabolic control in a solid mammalian organ.

Integration of microRNA miR-122 in hepatic circadian gene expression.

In liver, most metabolic pathways are under circadian control, and hundreds of protein-encoding genes are thus transcribed in a cyclic fashion. Here we show that rhythmic transcription extends to the locus specifying miR-122, a highly abundant, hepatocyte-specific microRNA. Genetic loss-of-function and gain-of-function experiments have identified the orphan nuclear receptor REV-ERBalpha as the major circadian regulator of mir-122 transcription. Although due to its long half-life mature miR-122 accumulates at nearly constant rates throughout the day, this miRNA is tightly associated with control mechanisms governing circadian gene expression. Thus, the knockdown of miR-122 expression via an antisense oligonucleotide (ASO) strategy resulted in the up- and down-regulation of hundreds of mRNAs, of which a disproportionately high fraction accumulates in a circadian fashion. miR-122 has previously been linked to the regulation of cholesterol and lipid metabolism. The transcripts associated with these pathways indeed show the strongest time point-specific changes upon miR-122 depletion. The identification of Pparbeta/delta and the peroxisome proliferator-activated receptor alpha (PPARalpha) coactivator Smarcd1/Baf60a as novel miR-122 targets suggests an involvement of the circadian metabolic regulators of the PPAR family in miR-122-mediated metabolic control.

Robust synchronization of coupled circadian and cell cycle oscillators in single mammalian cells.

Circadian cycles and cell cycles are two fundamental periodic processes with a period in the range of 1 day. Consequently, coupling between such cycles can lead to synchronization. Here, we estimated the mutual interactions between the two oscillators by time-lapse imaging of single mammalian NIH3T3 fibroblasts during several days. The analysis of thousands of circadian cycles in dividing cells clearly indicated that both oscillators tick in a 1:1 mode-locked state, with cell divisions occurring tightly 5 h before the peak in circadian Rev-Erbα-YFP reporter expression. In principle, such synchrony may be caused by either unidirectional or bidirectional coupling. While gating of cell division by the circadian cycle has been most studied, our data combined with stochastic modeling unambiguously show that the reverse coupling is predominant in NIH3T3 cells. Moreover, temperature, genetic, and pharmacological perturbations showed that the two interacting cellular oscillators adopt a synchronized state that is highly robust over a wide range of parameters. These findings have implications for circadian function in proliferative tissues, including epidermis, immune cells, and cancer.

CAVIN-3 regulates circadian period length and PER:CRY protein abundance and interactions.

In mammals, transcriptional autorepression by Period (PER) and Cryptochrome (CRY) protein complexes is essential for the generation of circadian rhythms. We have identified CAVIN-3 as a new, cytoplasmic PER2-interacting protein influencing circadian clock properties. Thus, CAVIN-3 loss- and gain-of-function shortened and lengthened, respectively, the circadian period in fibroblasts and affected PER:CRY protein abundance and interaction. While depletion of protein kinase Cδ (PKCδ), a known partner of CAVIN-3, had little effect on circadian gene expression, CAVIN-3 required the PKCδ-binding site to exert its effect on period length. This suggests the involvement of yet uncharacterized protein kinases. Finally, CAVIN-3 activity in circadian gene expression was independent of caveolae.

Diurnal control of iron responsive element containing mRNAs through iron regulatory proteins IRP1 and IRP2 is mediated by feeding rhythms.

BACKGROUND: Cellular iron homeostasis is regulated by iron regulatory proteins (IRP1 and IRP2) that sense iron levels (and other metabolic cues) and modulate mRNA translation or stability via interaction with iron regulatory elements (IREs). IRP2 is viewed as the primary regulator in the liver, yet our previous datasets showing diurnal rhythms for certain IRE-containing mRNAs suggest a nuanced temporal control mechanism. The purpose of this study is to gain insights into the daily regulatory dynamics across IRE-bearing mRNAs, specific IRP involvement, and underlying systemic and cellular rhythmicity cues in mouse liver. RESULTS: We uncover high-amplitude diurnal oscillations in the regulation of key IRE-containing transcripts in the liver, compatible with maximal IRP activity at the onset of the dark phase. Although IRP2 protein levels also exhibit some diurnal variations and peak at the light-dark transition, ribosome profiling in IRP2-deficient mice reveals that maximal repression of target mRNAs at this timepoint still occurs. We further find that diurnal regulation of IRE-containing mRNAs can continue in the absence of a functional circadian clock as long as feeding is rhythmic. CONCLUSIONS: Our findings suggest temporally controlled redundancy in IRP activities, with IRP2 mediating regulation of IRE-containing transcripts in the light phase and redundancy, conceivably with IRP1, at dark onset. Moreover, we highlight the significance of feeding-associated signals in driving rhythmicity. Our work highlights the dynamic nature and regulatory complexity in a metabolic pathway that had previously been considered well-understood.

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.

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.

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.

REV-ERBalpha participates in circadian SREBP signaling and bile acid homeostasis.

In mammals, many aspects of behavior and physiology, and in particular cellular metabolism, are coordinated by the circadian timing system. Molecular clocks are thought to rely on negative feedback loops in clock gene expression that engender oscillations in the accumulation of transcriptional regulatory proteins, such as the orphan receptor REV-ERBalpha. Circadian transcription factors then drive daily rhythms in the expression of clock-controlled output genes, for example genes encoding enzymes and regulators of cellular metabolism. To gain insight into clock output functions of REV-ERBalpha, we carried out genome-wide transcriptome profiling experiments with liver RNA from wild-type mice, Rev-erbalpha knock-out mice, or REV-ERBalpha overexpressing mice. On the basis of these genetic loss- and gain-of-function experiments, we concluded that REV-ERBalpha participates in the circadian modulation of sterol regulatory element-binding protein (SREBP) activity, and thereby in the daily expression of SREBP target genes involved in cholesterol and lipid metabolism. This control is exerted via the cyclic transcription of Insig2, encoding a trans-membrane protein that sequesters SREBP proteins to the endoplasmic reticulum membranes and thereby interferes with the proteolytic activation of SREBPs in Golgi membranes. REV-ERBalpha also participates in the cyclic expression of cholesterol-7alpha-hydroxylase (CYP7A1), the rate-limiting enzyme in converting cholesterol to bile acids. Our findings suggest that this control acts via the stimulation of LXR nuclear receptors by cyclically produced oxysterols. In conclusion, our study suggests that rhythmic cholesterol and bile acid metabolism is not just driven by alternating feeding-fasting cycles, but also by REV-ERBalpha, a component of the circadian clockwork circuitry.

Recording of Diurnal Gene Expression in Peripheral Organs of Mice Using the RT-Biolumicorder.

There is high interest in investigating the daily dynamics of gene expression in mammalian organs, for example, in liver. Such studies help to elucidate how and with what kinetics peripheral clocks integrate circadian signals from the suprachiasmatic nucleus, which harbors the circadian master pacemaker, with other systemic and environmental cues, such as those associated with feeding and hormones. Organ sampling around the clock, followed by the analysis of RNA and/or proteins, is the most commonly used procedure in assessing rhythmic gene expression. However, this method requires large cohorts of animals and is only applicable to behaviorally rhythmic animals whose phases are known. Real-time recording of gene expression rhythms using luciferase reporters has emerged as a powerful method to acquire continuous, high-resolution datasets from freely moving individual mice. Here, we share our experience and protocols with this technique, using the RT-Biolumicorder setup.

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.

Genome-wide oscillation of transcription in yeast.

Yeast cells growing under continuous conditions at high cellular density employ a robust metabolic cycle for energy generation in which a respiratory burst alternates with a non-respiratory, reductive phase. Two related studies have recently shown that global transcriptional co-regulation of genes defines the phases of this metabolic network in time and synchronizes cell division with metabolism. The finding that many fundamental and diverse cellular processes can be coordinated by global mRNA oscillations implies the existence of a more widespread metabolic clock that might also be present in higher eukaryotes.