Mitochondrial Ribosome Profiling Reveals Rapid and Dynamic Translation Regulation During Yeast Mitochondrial Biogenesis

AbstractIn addition to their use in relieving the symptoms of various diseases, ketogenic diets (KDs) have also been adopted by healthy individuals to prevent being overweight. Herein, we reported that prolonged KD exposure induced cardiac fibrosis. In rats, KD or frequent deep fasting decreased mitochondrial biogenesis, reduced cell respiration, and increased cardiomyocyte apoptosis and cardiac fibrosis. Mechanistically, increased levels of the ketone body β-hydroxybutyrate (β-OHB), an HDAC2 inhibitor, promoted histone acetylation of the Sirt7 promoter and activated Sirt7 transcription. This in turn inhibited the transcription of mitochondrial ribosome-encoding genes and mitochondrial biogenesis, leading to cardiomyocyte apoptosis and cardiac fibrosis. Exogenous β-OHB administration mimicked the effects of a KD in rats. Notably, increased β-OHB levels and SIRT7 expression, decreased mitochondrial biogenesis, and increased cardiac fibrosis were detected in human atrial fibrillation heart tissues. Our results highlighted the unknown detrimental effects of KDs and provided insights into strategies for preventing cardiac fibrosis in patients for whom KDs are medically necessary.

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Human Cytomegalovirus Infection Upregulates the Mitochondrial Transcription and Translation Machineries

mBio ◽

10.1128/mbio.00029-16 ◽

2016 ◽

Vol 7 (2) ◽

Cited By ~ 17

Author(s):

S. Karniely ◽

M. P. Weekes ◽

R. Antrobus ◽

J. Rorbach ◽

L. van Haute ◽

...

Keyword(s):

Tumor Cells ◽

Human Cytomegalovirus ◽

Mitochondrial Biogenesis ◽

Ribosome Biogenesis ◽

Hcmv Infection ◽

Aerobic Glycolysis ◽

Tricarboxylic Acid Cycle ◽

Mitochondrial Translation ◽

Mitochondrial Transcription ◽

Mitochondrial Ribosome

ABSTRACT Infection with human cytomegalovirus (HCMV) profoundly affects cellular metabolism. Like in tumor cells, HCMV infection increases glycolysis, and glucose carbon is shifted from the mitochondrial tricarboxylic acid cycle to the biosynthesis of fatty acids. However, unlike in many tumor cells, where aerobic glycolysis is accompanied by suppression of mitochondrial oxidative phosphorylation, HCMV induces mitochondrial biogenesis and respiration. Here, we affinity purified mitochondria and used quantitative mass spectrometry to determine how the mitochondrial proteome changes upon HCMV infection. We found that the mitochondrial transcription and translation systems are induced early during the viral replication cycle. Specifically, proteins involved in biogenesis of the mitochondrial ribosome were highly upregulated by HCMV infection. Inhibition of mitochondrial translation with chloramphenicol or knockdown of HCMV-induced ribosome biogenesis factor MRM3 abolished the HCMV-mediated increase in mitochondrially encoded proteins and significantly impaired viral growth under bioenergetically restricting conditions. Our findings demonstrate how HCMV manipulates mitochondrial biogenesis to support its replication. IMPORTANCE Human cytomegalovirus (HCMV), a betaherpesvirus, is a leading cause of morbidity and mortality during congenital infection and among immunosuppressed individuals. HCMV infection significantly changes cellular metabolism. Akin to tumor cells, in HCMV-infected cells, glycolysis is increased and glucose carbon is shifted from the tricarboxylic acid cycle to fatty acid biosynthesis. However, unlike in tumor cells, HCMV induces mitochondrial biogenesis even under aerobic glycolysis. Here, we have affinity purified mitochondria and used quantitative mass spectrometry to determine how the mitochondrial proteome changes upon HCMV infection. We find that the mitochondrial transcription and translation systems are induced early during the viral replication cycle. Specifically, proteins involved in biogenesis of the mitochondrial ribosome were highly upregulated by HCMV infection. Inhibition of mitochondrial translation with chloramphenicol or knockdown of HCMV-induced ribosome biogenesis factor MRM3 abolished the HCMV-mediated increase in mitochondrially encoded proteins and significantly impaired viral growth. Our findings demonstrate how HCMV manipulates mitochondrial biogenesis to support its replication.

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Inferring translational heterogeneity from ribosome profiling data

10.1101/866582 ◽

2019 ◽

Author(s):

Pedro do Couto Bordignon ◽

Sebastian Pechmann

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein Biosynthesis ◽

Ribosome Profiling ◽

Translation Regulation ◽

Messenger Rnas ◽

Translation Elongation ◽

Cellular Homeostasis ◽

Ribosome Density ◽

Experimental Variability ◽

Underlying Mechanisms

Translation of messenger RNAs into proteins by the ribosome is the most important step of protein biosynthesis. Accordingly, translation is tightly controlled and heavily regulated to maintain cellular homeostasis. Ribosome profiling (Ribo-seq) has revolutionized the study of translation by revealing many of its underlying mechanisms. However, equally many aspects of translation remain mysterious, in part also due to persisting challenges in the interpretation of data obtained from Ribo-seq experiments. Here, we show that some of the variability observed in Ribo-seq data has biological origins and reflects programmed heterogeneity of translation. To systematically identify sequences that are differentially translated (DT) across mRNAs beyond what can be attributed to experimental variability, we performed a comparative analysis of Ribo-seq data from Saccharomyces cerevisiae and derived a consensus ribosome density profile that reflects consistent signals in individual experiments. Remarkably, the thus identified DT sequences link to mechanisms known to regulate translation elongation and are enriched in genes important for protein and organelle biosynthesis. Our results thus highlight examples of translational heterogeneity that are encoded in the genomic sequences and tuned to optimizing cellular homeostasis. More generally, our work highlights the power of Ribo-seq to understand the complexities of translation regulation.

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Generally applicable transcriptome-wide analysis of translational efficiency using anota2seq

10.1101/106922 ◽

2017 ◽

Cited By ~ 3

Author(s):

Christian Oertlin ◽

Julie Lorent ◽

Valentina Gandin ◽

Carl Murie ◽

Laia Masvidal ◽

...

Keyword(s):

Gene Expression ◽

Analytical Methods ◽

Regulation Of Gene Expression ◽

Mrna Translation ◽

Ribosome Profiling ◽

Translation Regulation ◽

Translational Efficiency ◽

Mrna Levels ◽

Dependent Manner ◽

Mtor Inhibition

ABSTRACTmRNA translation plays an evolutionarily conserved role in homeostasis and when dysregulated results in various disorders. Optimal and universally applicable analytical methods to study transcriptome-wide changes in translational efficiency are therefore critical for understanding the complex role of translation regulation under physiological and pathological conditions. Techniques used to interrogate translatomes, including polysome- and ribosome-profiling, require adjustment for changes in total mRNA levels to capture bona fide alterations in translational efficiency. Herein, we present the anota2seq algorithm for such analysis using data from ribosome- or polysome-profiling quantified by DNA-microarrays or RNA sequencing, which outperforms current methods for identification of changes in translational efficiency. In contrast to available analytical methods, anota2seq also allows capture of an underappreciated mode for regulation of gene expression whereby translation acts as a buffering mechanism which maintains constant protein levels despite fluctuations in mRNA levels (“translational buffering”). Application of anota2seq shows that insulin affects gene expression at multiple levels, in a largely mTOR-dependent manner. Moreover, insulin induces levels of a subset of mRNAs independently of mTOR that undergo translational buffering upon mTOR inhibition. Thus, the universal anota2seq algorithm allows efficient and hitherto unprecedented interrogation of translatomes and enables studies of translational buffering which represents an unexplored mechanism for regulating of gene expression.

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RiboDiff: Detecting Changes of Translation Efficiency from Ribosome Footprints

10.1101/017111 ◽

2015 ◽

Cited By ~ 3

Author(s):

Yi Zhong ◽

Theofanis Karaletsos ◽

Philipp Drewe ◽

Vipin Thankam T Sreedharan ◽

David Kuo ◽

...

Keyword(s):

Linear Models ◽

Protein Translation ◽

Ribosome Profiling ◽

Translation Regulation ◽

Analysis Tool ◽

Translation Efficiency ◽

Case Control Studies ◽

Statistical Framework ◽

Experimental Treatments ◽

Over Dispersion

Motivation: Deep sequencing based ribosome footprint profiling can provide novel insights into the regulatory mechanisms of protein translation. However, the observed ribosome profile is fundamentally confounded by transcriptional activity. In order to decipher principles of translation regulation, tools that can reliably detect changes in translation efficiency in case-control studies are needed. Results: We present a statistical framework and analysis tool, RiboDiff, to detect genes with changes in translation efficiency across experimental treatments. RiboDiff uses generalized linear models to estimate the over-dispersion of RNA-Seq and ribosome profiling measurements separately, and performs a statistical test for differential translation efficiency using both mRNA abundance and ribosome occupancy. Availability: Source code and documentation are available at http://github.com/ratschlab/ribodiff. Supplementary Material can be found at http://bioweb.me/ribo.

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Autonomous functionality of an upstream open reading frame in polycistronic mammalian mRNAs

10.1101/325571 ◽

2018 ◽

Author(s):

Shohei Kitano ◽

Gabriel Pratt ◽

Keizo Takao ◽

Yasunori Aizawa

Keyword(s):

Brain Regions ◽

Ribosome Profiling ◽

Open Reading Frames ◽

Upstream Open Reading Frame ◽

Translation Regulation ◽

Reading Frame ◽

Eukaryotic Translation ◽

Upstream Open Reading Frames ◽

Human And Mouse ◽

Reading Frames

SUMMARYUpstream open reading frames (uORFs) are established as cis-acting elements for eukaryotic translation of annotated ORFs (anORFs) located on the same mRNAs. Here, we identified a mammalian uORF with functions that are independent from anORF translation regulation. Bioinformatics screening using ribosome profiling data of human and mouse brains yielded 308 neurologically vital genes from which anORF and uORFs are polycistronically translated in both species. Among them, Arhgef9 contains a uORF named SPICA, which is highly conserved among vertebrates and stably translated only in specific brain regions of mice. Disruption of SPICA translation by ATG-to-TAG substitutions did not perturb translation or function of its anORF product, collybistin. SPICA-null mice displayed abnormal maternal reproductive performance and enhanced anxiety-like behavior, characteristic of ARHGEF9-associated neurological disorders. This study demonstrates that mammalian uORFs can be independent genetic units, revising the prevailing dogma of the monocistronic gene in mammals, and even eukaryotes.

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RNA G-quadruplexes mark repressive upstream open reading frames in human mRNAs

10.1101/223073 ◽

2017 ◽

Cited By ~ 1

Author(s):

Pierre Murat ◽

Giovanni Marsico ◽

Barbara Herdy ◽

Avazeh Ghanbarian ◽

Guillem Portella ◽

...

Keyword(s):

Secondary Structures ◽

Ribosome Profiling ◽

Open Reading Frames ◽

Untranslated Regions ◽

Translation Regulation ◽

Physical Interaction ◽

Protein Coding ◽

Upstream Open Reading Frames ◽

Nucleotide Resolution ◽

Reading Frames

ABSTRACTRNA secondary structures in the 5’ untranslated regions (UTRs) of mRNAs have been characterised as key determinants of translation initiation. However the role of non-canonical secondary structures, such as RNA G-quadruplexes (rG4s), in modulating translation of human mRNAs and the associated mechanisms remain largely unappreciated. Here we use a ribosome profiling strategy to investigate the translational landscape of human mRNAs with structured 5’ untranslated regions (5’-UTR). We found that inefficiently translated mRNAs, containing rG4-forming sequences in their 5’-UTRs, have an accumulation of ribosome footprints in their 5’-UTRs. We show that rG4-forming sequences are determinants of 5’-UTR translation, suggesting that the folding of rG4 structures thwarts the translation of protein coding sequences (CDS) by stimulating the translation of repressive upstream open reading frames (uORFs). To support our model, we demonstrate that depletion of two rG4s-specialised DEAH-box helicases, DHX36 and DHX9, shifts translation towards rG4-containing uORFs reducing the translation of selected transcripts comprising proto-oncogenes, transcription factors and epigenetic regulators. Transcriptome-wide identification of DHX9 binding sites using individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) demonstrate that translation regulation is mediated through direct physical interaction between the helicase and its rG4 substrate. Our findings unveil a previously unknown role for non-canonical structures in governing 5’-UTR translation and suggest that the interaction of helicases with rG4s could be considered as a target for future therapeutic intervention.

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Dynamic translation regulation inCaulobactercell cycle control

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1614795113 ◽

2016 ◽

Vol 113 (44) ◽

pp. E6859-E6867 ◽

Cited By ~ 32

Author(s):

Jared M. Schrader ◽

Gene-Wei Li ◽

W. Seth Childers ◽

Adam M. Perez ◽

Jonathan S. Weissman ◽

...

Keyword(s):

Cell Cycle ◽

Translational Control ◽

Transcriptional Control ◽

Relative Rate ◽

Ribosome Profiling ◽

Translation Regulation ◽

Translational Efficiency ◽

Specific Cell ◽

Control Of Gene Expression ◽

Regulatory Pathways

Progression of theCaulobactercell cycle requires temporal and spatial control of gene expression, culminating in an asymmetric cell division yielding distinct daughter cells. To explore the contribution of translational control, RNA-seq and ribosome profiling were used to assay global transcription and translation levels of individual genes at six times over the cell cycle. Translational efficiency (TE) was used as a metric for the relative rate of protein production from each mRNA. TE profiles with similar cell cycle patterns were found across multiple clusters of genes, including those in operons or in subsets of operons. Collections of genes associated with central cell cycle functional modules (e.g., biosynthesis of stalk, flagellum, or chemotaxis machinery) have consistent but different TE temporal patterns, independent of their operon organization. Differential translation of operon-encoded genes facilitates precise cell cycle-timing for the dynamic assembly of multiprotein complexes, such as the flagellum and the stalk and the correct positioning of regulatory proteins to specific cell poles. The cell cycle-regulatory pathways that produce specific temporal TE patterns are separate from—but highly coordinated with—the transcriptional cell cycle circuitry, suggesting that the scheduling of translational regulation is organized by the same cyclical regulatory circuit that directs the transcriptional control of theCaulobactercell cycle.

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Ribosome Profiling in Archaea Reveals Leaderless Translation, Novel Translational Initiation Sites, and Ribosome Pausing at Single Codon Resolution

10.1101/2020.02.04.934349 ◽

2020 ◽

Cited By ~ 1

Author(s):

Diego Rivera Gelsinger ◽

Emma Dallon ◽

Rahul Reddy ◽

Fuad Mohammad ◽

Allen R. Buskirk ◽

...

Keyword(s):

Large Scale ◽

Scale Effects ◽

Ribosome Profiling ◽

Translation Regulation ◽

Cellular Functions ◽

Translational Initiation ◽

The Third ◽

Intergenic Regions ◽

Ribosome Pausing ◽

First Time

ABSTRACTHigh-throughput methods, such as ribosome profiling, have revealed the complexity of translation regulation in Bacteria and Eukarya with large-scale effects on cellular functions. In contrast, the translational landscape in Archaea remains mostly unexplored. Here, we developed ribosome profiling in a model archaeon, Haloferax volcanii, elucidating, for the first time, the translational landscape of a representative of the third domain of life. We determined the ribosome footprint of H. volcanii to be comparable in size to that of the Eukarya. We linked footprint lengths to initiating and elongating states of the ribosome on leadered transcripts, operons, and on leaderless transcripts, the latter representing 70% of H. volcanii transcriptome. We manipulated ribosome activity with translation inhibitors to reveal ribosome pausing at specific codons. Lastly, we found that the drug harringtonine arrested ribosomes at initiation sites in this archaeon. This drug treatment allowed us to confirm known translation initiation sites and also reveal putative novel initiation sites in intergenic regions and within genes. Ribosome profiling revealed an uncharacterized complexity of translation in this archaeon with bacteria-like, eukarya-like, and potentially novel translation mechanisms. These mechanisms are likely to be functionally essential and to contribute to an expanded proteome with regulatory roles in gene expression.

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Maturation of selected human mitochondrial tRNAs requires deadenylation

eLife ◽

10.7554/elife.27596 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 33

Author(s):

Sarah F Pearce ◽

Joanna Rorbach ◽

Lindsey Van Haute ◽

Aaron R D’Souza ◽

Pedro Rebelo-Guiomar ◽

...

Keyword(s):

Quality Control ◽

Life Cycle ◽

Oxidative Phosphorylation ◽

Ribosome Profiling ◽

Oxidative Phosphorylation System ◽

A Genome ◽

Mitochondrial Ribosome ◽

Rna Quality Control ◽

Non Coding Rnas ◽

Precursor Rna

Human mitochondria contain a genome (mtDNA) that encodes essential subunits of the oxidative phosphorylation system. Expression of mtDNA entails multi-step maturation of precursor RNA. In other systems, the RNA life cycle involves surveillance mechanisms, however, the details of RNA quality control have not been extensively characterised in human mitochondria. Using a mitochondrial ribosome profiling and mitochondrial poly(A)-tail RNA sequencing (MPAT-Seq) assay, we identify the poly(A)-specific exoribonuclease PDE12 as a major factor for the quality control of mitochondrial non-coding RNAs. The lack of PDE12 results in a spurious polyadenylation of the 3’ ends of the mitochondrial (mt-) rRNA and mt-tRNA. While the aberrant adenylation of 16S mt-rRNA did not affect the integrity of the mitoribosome, spurious poly(A) additions to mt-tRNA led to reduced levels of aminoacylated pool of certain mt-tRNAs and mitoribosome stalling at the corresponding codons. Therefore, our data uncover a new, deadenylation-dependent mtRNA maturation pathway in human mitochondria.

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