Monitoring translation in all reading frames downstream of weak stop codons provides mechanistic insights into the impact of nucleotide and cellular contexts.

A stop codon entering the ribosome A-site is normally decoded by release factors that induce release of the polypeptide. Certain factors influence the efficiency of the termination which is in competition with elongation in either the same (readthrough) or an alternative (frameshifting) reading frame. To gain insight into the competition between these processes, we monitored translation in parallel from all three reading frames downstream of stop codons while changing the nucleotide context of termination sites or altering cellular conditions (polyamine levels). We found that P-site codon identity can have a major impact on the termination efficiency of the OPRL1 stop signal, whereas for the OAZ1 ORF1 stop signal, the P-site codon mainly influences the reading frame of non-terminating ribosomes. Changes to polyamine levels predominantly influence the termination efficiency of the OAZ1 ORF1 stop signal. In contrast, increasing polyamine levels stimulate readthrough of the OPRL1 stop signal by enhancing near-cognate decoding rather than by decreasing termination efficiency. Thus, by monitoring the four competing processes occurring at stop codons we were able to determine which is the most significantly affected upon perturbation. This approach may be useful for the interrogation of other recoding phenomena where alternative decoding processes compete with standard decoding.

Lack of evidence for ribosomal frameshifting in ATP7B mRNA decoding.

The research article describing the discovery of ribosomal frameshifting in the bacterial CopA gene also reported the occurrence of frameshifting in the expression of the human ortholog ATP7B based on assays using dual luciferase reporters. An examination of the publicly available ribosome profiling data and the phylogenetic analysis of the proposed frameshifting site cast doubt on the validity of this claim and prompted us to reexamine the evidence. We observed similar apparent frameshifting efficiencies as the original authors using the same type of vector that synthesizes both luciferases as a single polyprotein. However, we noticed anomalously low absolute luciferase activities from the N-terminal reporter that suggests interference of reporter activity or levels by the ATP7B test cassette. When we tested the same proposed ATP7B frameshifting cassette in a more recently developed reporter system in which the reporters are released without being included in a polyprotein, no frameshifting was detected above background levels.

Mitochondrial complex IV defects induce metabolic and signaling perturbations that expose potential vulnerabilities in HCT116 cells.

Mutations in genes encoding cytochrome c oxidase (mitochondrial complex IV) subunits and assembly factors [e.g., synthesis of cytochrome c oxidase 2 (SCO2)] are linked to severe metabolic syndromes. Notwithstanding that SCO2 is under transcriptional control of tumor suppressor p53, the role of mitochondrial complex IV dysfunction in cancer metabolism remains obscure. Herein, we demonstrate that the loss of SCO2 in HCT116 colorectal cancer cells leads to significant metabolic and signaling perturbations. Specifically, abrogation of SCO2 increased NAD+ regenerating reactions and decreased glucose oxidation through citric acid cycle while enhancing pyruvate carboxylation. This was accompanied by a reduction in amino acid levels and the accumulation of lipid droplets. In addition, SCO2 loss resulted in hyperactivation of the insulin-like growth factor 1 receptor (IGF1R)/AKT axis with paradoxical downregulation of mTOR signaling, which was accompanied by increased AMP-activated kinase activity. Accordingly, abrogation of SCO2 expression appears to increase the sensitivity of cells to IGF1R and AKT, but not mTOR inhibitors. Finally, the loss of SCO2 was associated with reduced proliferation and enhanced migration of HCT116 cells. Collectively, herein we describe potential adaptive signaling and metabolic perturbations triggered by mitochondrial complex IV dysfunction.

Identification and characterization of hippuristanol-resistant mutants reveals eIF4A1 dependencies within mRNA 5' leader regions.

Hippuristanol (Hipp) is a natural product that selectively inhibits protein synthesis by targeting eukaryotic initiation factor (eIF) 4A, a DEAD-box RNA helicase required for ribosome recruitment to mRNA templates. Hipp binds to the carboxyl-terminal domain of eIF4A, locks it in a closed conformation, and inhibits its RNA binding. The dependencies of mRNAs for eIF4A during initiation is contingent on the degree of secondary structure within their 5' leader region. Interest in targeting eIF4A therapeutically in cancer and viral-infected settings stems from the dependencies that certain cellular (e.g. pro-oncogenic, pro-survival) and viral mRNAs show towards eIF4A. Using a CRISPR/Cas9-based variomics screen, we identify functional EIF4A1 Hipp-resistant alleles, which in turn allowed us to link the translation-inhibitory and cytotoxic properties of Hipp to eIF4A1 target engagement. Genome-wide translational profiling in the absence or presence of Hipp were undertaken and our validation studies provided insight into the structure-activity relationships of eIF4A-dependent mRNAs. We find that mRNA 5' leader length, overall secondary structure and cytosine content are defining features of Hipp-dependent mRNAs.

Rocaglates Induce Gain-of-Function Alterations to eIF4A and eIF4F.

Rocaglates are a diverse family of biologically active molecules that have gained tremendous interest in recent years due to their promising activities in pre-clinical cancer studies. As a result, this family of compounds has been significantly expanded through the development of efficient synthetic schemes. However, it is unknown whether all of the members of the rocaglate family act through similar mechanisms of action. Here, we present a comprehensive study comparing the biological activities of >200 rocaglates to better understand how the presence of different chemical entities influences their biological activities. Through this, we find that most rocaglates preferentially repress the translation of mRNAs containing purine-rich 5' leaders, but certain rocaglates lack this bias in translation repression. We also uncover an aspect of rocaglate mechanism of action in which the pool of translationally active eIF4F is diminished due to the sequestration of the complex onto RNA.

In Vivo RNAi-Mediated eIF3m Knockdown Affects Ribosome Biogenesis and Transcription but Has Limited Impact on mRNA-Specific Translation.

Translation is an essential biological process, and dysregulation is associated with a range of diseases including ribosomopathies, diabetes, and cancer. Here, we examine translation dysregulation in vivo using RNAi to knock down the m-subunit of the translation initiation factor eIF3 in the mouse liver. Transcriptome sequencing, ribosome profiling, whole proteome, and phosphoproteome analyses show that eIF3m deficiency leads to the transcriptional response and changes in cellular translation that yield few detectable differences in the translation of particular mRNAs. The transcriptional response fell into two main categories: ribosome biogenesis (increased transcription of ribosomal proteins) and cell metabolism (alterations in lipid, amino acid, nucleic acid, and drug metabolism). Analysis of ribosome biogenesis reveals inhibition of rRNA processing, highlighting decoupling of rRNA synthesis and ribosomal protein gene transcription in response to eIF3m knockdown. Interestingly, a similar reduction in eIF3m protein levels is associated with induction of the mTOR pathway in vitro but not in vivo. Overall, this work highlights the utility of a RNAi-based in vivo approach for studying the regulation of mammalian translation in vivo.

Cellular Gene Expression during Hepatitis C Virus Replication as Revealed by Ribosome Profiling.

BACKGROUND: Hepatitis C virus (HCV) infects human liver hepatocytes, often leading to liver cirrhosis and hepatocellular carcinoma (HCC). It is believed that chronic infection alters host gene expression and favors HCC development. In particular, HCV replication in Endoplasmic Reticulum (ER) derived membranes induces chronic ER stress. How HCV replication affects host mRNA translation and transcription at a genome wide level is not yet known. METHODS: We used Riboseq (Ribosome Profiling) to analyze transcriptome and translatome changes in the Huh-7.5 hepatocarcinoma cell line replicating HCV for 6 days. RESULTS: Established viral replication does not cause global changes in host gene expression-only around 30 genes are significantly differentially expressed. Upregulated genes are related to ER stress and HCV replication, and several regulated genes are known to be involved in HCC development. Some mRNAs (PPP1R15A/GADD34, DDIT3/CHOP, and TRIB3) may be subject to upstream open reading frame (uORF) mediated translation control. Transcriptional downregulation mainly affects mitochondrial respiratory chain complex core subunit genes. CONCLUSION: After establishing HCV replication, the lack of global changes in cellular gene expression indicates an adaptation to chronic infection, while the downregulation of mitochondrial respiratory chain genes indicates how a virus may further contribute to cancer cell-like metabolic reprogramming ("Warburg effect") even in the hepatocellular carcinoma cells used here.

Magnesium-sensitive upstream ORF controls PRL phosphatase expression to mediate energy metabolism.

Phosphatases of regenerating liver (PRL-1, PRL-2, and PRL-3, also known as PTP4A1, PTP4A2, and PTP4A3) control magnesium homeostasis through an association with the CNNM magnesium transport regulators. Although high PRL levels have been linked to cancer progression, regulation of their expression is poorly understood. Here we show that modulating intracellular magnesium levels correlates with a rapid change of PRL expression by a mechanism involving its 5'UTR mRNA region. Mutations or CRISPR-Cas9 targeting of the conserved upstream ORF present in the mRNA leader derepress PRL protein synthesis and attenuate the translational response to magnesium levels. Mechanistically, magnesium depletion reduces intracellular ATP but up-regulates PRL protein expression via activation of the AMPK/mTORC2 pathway, which controls cellular energy status. Hence, altered PRL-2 expression leads to metabolic reprogramming of the cells. These findings uncover a magnesium-sensitive mechanism controlling PRL expression, which plays a role in cellular bioenergetics.

The GWIPS-viz Browser.

GWIPS-viz is a publicly available browser that provides Genome Wide Information on Protein Synthesis through the visualization of ribosome profiling data. Ribosome profiling (Ribo-seq) is a high-throughput technique which isolates fragments of messenger RNA that are protected by the ribosome. The alignment of the ribosome-protected fragments or footprint sequences to the corresponding reference genome and their visualization using GWIPS-viz allows for unique insights into the genome loci that are expressed as potentially translated RNA. The GWIPS-viz browser hosts both Ribo-seq data and corresponding mRNA-seq data from publicly available studies across a number of genomes, avoiding the need for computational processing on the user side. Since its initial publication in 2014, over 1885 tracks have been produced across 24 genomes. This unit describes the navigation of the GWIPS-viz genome browser, the uploading of custom tracks, and the downloading of the Ribo-seq/mRNA-seq alignment data. © 2018 by John Wiley & Sons, Inc.

Positioning Europe for the EPITRANSCRIPTOMICS challenge.

The genetic alphabet consists of the four letters: C, A, G, and T in DNA and C,A,G, and U in RNA. Triplets of these four letters jointly encode 20 different amino acids out of which proteins of all organisms are built. This system is universal and is found in all kingdoms of life. However, bases in DNA and RNA can be chemically modified. In DNA, around 10 different modifications are known, and those have been studied intensively over the past 20 years. Scientific studies on DNA modifications and proteins that recognize them gave rise to the large field of epigenetic and epigenomic research. The outcome of this intense research field is the discovery that development, ageing, and stem-cell dependent regeneration but also several diseases including cancer are largely controlled by the epigenetic state of cells. Consequently, this research has already led to the first FDA approved drugs that exploit the gained knowledge to combat disease. In recent years, the ~150 modifications found in RNA have come to the focus of intense research. Here we provide a perspective on necessary and expected developments in the fast expanding area of RNA modifications, termed epitranscriptomics.

Translation control of mRNAs encoding mammalian translation initiation factors.

Eukaryotic cells evolved highly complex and accurate protein synthesis machinery that is finely tuned by various signaling pathways. Dysregulation of translation is a hallmark of many diseases, including cancer, and thus pharmacological approaches to modulate translation become very promising. While there has been much progress in our understanding of mammalian mRNA-specific translation control, surprisingly, relatively little is known about whether and how the protein components of the translation machinery shape translation of their own mRNAs. Here we analyze mammalian mRNAs encoding components of the translation initiation machinery for potential regulatory features such as 5'TOP motifs, TISU motifs, poor start codon nucleotide context and upstream open reading frames.

AMD1 mRNA employs ribosome stalling as a mechanism for molecular memory formation.

In addition to acting as template for protein synthesis, messenger RNA (mRNA) often contains sensory sequence elements that regulate this process. Here we report a new mechanism that limits the number of complete protein molecules that can be synthesized from a single mRNA molecule of the human AMD1 gene encoding adenosylmethionine decarboxylase 1 (AdoMetDC). A small proportion of ribosomes translating AMD1 mRNA stochastically read through the stop codon of the main coding region. These readthrough ribosomes then stall close to the next in-frame stop codon, eventually forming a ribosome queue, the length of which is proportional to the number of AdoMetDC molecules that were synthesized from the same AMD1 mRNA. Once the entire spacer region between the two stop codons is filled with queueing ribosomes, the queue impinges upon the main AMD1 coding region halting its translation. Phylogenetic analysis suggests that this mechanism is highly conserved in vertebrates and existed in their common ancestor. We propose that this mechanism is used to count and limit the number of protein molecules that can be synthesized from a single mRNA template. It could serve to safeguard from dysregulated translation that may occur owing to errors in transcription or mRNA damage.

Stimulation of reverse transcriptase generated cDNAs with specific indels by template RNA structure: retrotransposon, dNTP balance, RT-reagent usage.

RNA dependent DNA-polymerases, reverse transcriptases, are key enzymes for retroviruses and retroelements. Their fidelity, including indel generation, is significant for their use as reagents including for deep sequencing. Here, we report that certain RNA template structures and G-rich sequences, ahead of diverse reverse transcriptases can be strong stimulators for slippage at slippage-prone template motif sequence 3' of such 'slippage-stimulatory' structures. Where slippage is stimulated, the resulting products have one or more additional base(s) compared to the corresponding template motif. Such structures also inhibit slippage-mediated base omission which can be more frequent in the absence of a relevant stem-loop. Slippage directionality, base insertion and omission, is sensitive to the relative concentration ratio of dNTPs specified by the RNA template slippage-prone sequence and its 5' adjacent base. The retrotransposon-derived enzyme TGIRT exhibits more slippage in vitro than the retroviral enzymes tested including that from HIV. Structure-mediated slippage may be exhibited by other polymerases and enrich gene expression. A cassette from Drosophila retrotransposon Dme1_chrX_2630566, a candidate for utilizing slippage for its GagPol synthesis, exhibits strong slippage in vitro. Given the widespread occurrence and importance of retrotransposons, systematic studies to reveal the extent of their functional utilization of RT slippage are merited.

Specific reverse transcriptase slippage at the HIV ribosomal frameshift sequence: potential implications for modulation of GagPol synthesis.

Synthesis of HIV GagPol involves a proportion of ribosomes translating a U6A shift site at the distal end of the gag gene performing a programmed -1 ribosomal frameshift event to enter the overlapping pol gene. In vitro studies here show that at the same shift motif HIV reverse transcriptase generates -1 and +1 indels with their ratio being sensitive to the relative concentration ratio of dNTPs specified by the RNA template slippage-prone sequence and its 5' adjacent base. The GGG sequence 3' adjacent to the U6A shift/slippage site, which is important for ribosomal frameshifting, is shown here to limit reverse transcriptase base substitution and indel 'errors' in the run of A's in the product. The indels characterized here have either 1 more or less A, than the corresponding number of template U's. cDNA with 5 A's may yield novel Gag product(s), while cDNA with an extra base, 7 A's, may only be a minor contributor to GagPol polyprotein. Synthesis of a proportion of non-ribosomal frameshift derived GagPol would be relevant in efforts to identify therapeutically useful compounds that perturb the ratio of GagPol to Gag, and pertinent to the extent in which specific polymerase slippage is utilized in gene expression.

Low energy costs of F1Fo ATP synthase reversal in colon carcinoma cells deficient in mitochondrial complex IV.

Mitochondrial polarisation is paramount for a variety of cellular functions. Under ischemia, mitochondrial membrane potential (ΔΨm) and proton gradient (ΔpH) are maintained via a reversal of mitochondrial F1Fo ATP synthase (mATPase), which can rapidly deplete ATP and drive cells into energy crisis. We found that under normal conditions in cells with disassembled cytochrome c oxidase complex (COX-deficient HCT116), mATPase maintains ΔΨm at levels only 15-20% lower than in WT cells, and for this utilises relatively little ATP. For a small energy expenditure, mATPase enables mitochondrial ΔpH, protein import, Ca2+ turnover, and supports free radical detoxication machinery enlarged to protect the cells from oxidative damage. Whereas in COX-deficient cells the main source of ATP is glycolysis, the ΔΨm is still maintained upon inhibition of the adenine nucleotide translocators with bongkrekic acid and carboxyatractyloside, indicating that the role of ANTs is redundant, and matrix substrate level phosphorylation alone or in cooperation with ATP-Mg/Pi carriers can continuously support the mATPase activity. Intriguingly, we found that mitochondrial complex III is active, and it contributes not only to free radical production, but also to ΔΨm maintenance and energy budget of COX-deficient cells. Overall, this study demonstrates that F1Fo ATP synthase can support general mitochondrial and cellular functions, working in extremely efficient 'energy saving' reverse mode and flexibly recruiting free radical detoxication and ATP producing / transporting pathways.

Insights into the mechanisms of eukaryotic translation gained with ribosome profiling.

The development of Ribosome Profiling (RiboSeq) has revolutionized functional genomics. RiboSeq is based on capturing and sequencing of the mRNA fragments enclosed within the translating ribosome and it thereby provides a 'snapshot' of ribosome positions at the transcriptome wide level. Although the method is predominantly used for analysis of differential gene expression and discovery of novel translated ORFs, the RiboSeq data can also be a rich source of information about molecular mechanisms of polypeptide synthesis and translational control. This review will focus on how recent findings made with RiboSeq have revealed important details of the molecular mechanisms of translation in eukaryotes. These include mRNA translation sensitivity to drugs affecting translation initiation and elongation, the roles of upstream ORFs in response to stress, the dynamics of elongation and termination as well as details of intrinsic ribosome behavior on the mRNA after translation termination. As the RiboSeq method is still at a relatively early stage we will also discuss the implications of RiboSeq artifacts on data interpretation.

Systematic analysis of the PTEN 5' leader identifies a major AUU initiated proteoform.

Abundant evidence for translation within the 5' leaders of many human genes is rapidly emerging, especially, because of the advent of ribosome profiling. In most cases, it is believed that the act of translation rather than the encoded peptide is important. However, the wealth of available sequencing data in recent years allows phylogenetic detection of sequences within 5' leaders that have emerged under coding constraint and therefore allow for the prediction of functional 5' leader translation. Using this approach, we previously predicted a CUG-initiated, 173 amino acid N-terminal extension to the human tumour suppressor PTEN. Here, a systematic experimental analysis of translation events in the PTEN 5' leader identifies at least two additional non-AUG-initiated PTEN proteoforms that are expressed in most human cell lines tested. The most abundant extended PTEN proteoform initiates at a conserved AUU codon and extends the canonical AUG-initiated PTEN by 146 amino acids. All N-terminally extended PTEN proteoforms tested retain the ability to downregulate the PI3K pathway. We also provide evidence for the translation of two conserved AUG-initiated upstream open reading frames within the PTEN 5' leader that control the ratio of PTEN proteoforms.

The effect of heterogeneous Transcription Start Sites (TSS) on the translatome: implications for the mammalian cellular phenotype.

BACKGROUND: The genetic program, as manifested as the cellular phenotype, is in large part dictated by the cell's protein composition. Since characterisation of the proteome remains technically laborious it is attractive to define the genetic expression profile using the transcriptome. However, the transcriptional landscape is complex and it is unclear as to what extent it reflects the ribosome associated mRNA population (the translatome). This is particularly pertinent for genes using multiple transcriptional start sites (TSS) generating mRNAs with heterogeneous 5' transcript leaders (5'TL). Furthermore, the relative abundance of the TSS gene variants is frequently cell-type specific. Indeed, promoter switches have been reported in pathologies such as cancer. The consequences of this 5'TL heterogeneity within the transcriptome for the translatome remain unresolved. This is not a moot point because the 5'TL plays a key role in regulating mRNA recruitment onto polysomes. RESULTS: In this article, we have characterised both the transcriptome and translatome of the MCF7 (tumoural) and MCF10A (non-tumoural) cell lines. We identified ~550 genes exhibiting differential translation efficiency (TE). In itself, this is maybe not surprising. However, by focusing on genes exhibiting TSS heterogeneity we observed distinct differential promoter usage patterns in both the transcriptome and translatome. Only a minor fraction of these genes belonged to those exhibiting differential TE. Nonetheless, reporter assays demonstrated that the TSS variants impacted on the translational readout both quantitatively (the overall amount of protein expressed) and qualitatively (the nature of the proteins expressed). CONCLUSIONS: The results point to considerable and distinct cell-specific 5'TL heterogeneity within both the transcriptome and translatome of the two cell lines analysed. This observation is in-line with the ribosome filter hypothesis which posits that the ribosomal machine can selectively filter information from within the transcriptome. As such it cautions against the simple extrapolation transcriptome → proteome. Furthermore, polysomal occupancy of specific gene 5'TL variants may also serve as novel disease biomarkers.

Oxygen and glucose deprivation induces widespread alterations in mRNA translation within 20 minutes.

BACKGROUND: Oxygen and glucose metabolism play pivotal roles in many (patho)physiological conditions. In particular, oxygen and glucose deprivation (OGD) during ischemia and stroke results in extensive tissue injury and cell death. RESULTS: Using time-resolved ribosome profiling, we assess gene expression levels in a neural cell line, PC12, during the first hour of OGD. The most substantial alterations are seen to occur within the first 20 minutes of OGD. While transcription of only 100 genes is significantly altered during one hour of OGD, the translation response affects approximately 3,000 genes. This response involves reprogramming of initiation and elongation rates, as well as the stringency of start codon recognition. Genes involved in oxidative phosphorylation are most affected. Detailed analysis of ribosome profiles reveals salient alterations of ribosome densities on individual mRNAs. The mRNA-specific alterations include increased translation of upstream open reading frames, site-specific ribosome pauses, and production of alternative protein isoforms with amino-terminal extensions. Detailed analysis of ribosomal profiles also reveals six mRNAs with translated ORFs occurring downstream of annotated coding regions and two examples of dual coding mRNAs, where two protein products are translated from the same long segment of mRNA, but in two different frames. CONCLUSIONS: These findings uncover novel regulatory mechanisms of translational response to OGD in mammalian cells that are different from the classical pathways such as hypoxia inducible factor (HIF) signaling, while also revealing sophisticated organization of protein coding information in certain genes.