RPL3L-containing ribosomes modulate mitochondrial activity in the mammalian heart

ABSTRACTThe existence of naturally occurring ribosome heterogeneity is now a well-acknowledged phenomenon. However, whether this heterogeneity leads to functionally diverse ‘specialized ribosomes’ is still a controversial topic. Here, we explore the biological function of RPL3L, a ribosomal protein (RP) paralog of RPL3 that is exclusively expressed in muscle and heart tissues, by generating a viable homozygous Rpl3l knockout mouse strain. We identify a rescue mechanism in which, upon Rpl3l depletion, RPL3 becomes upregulated, yielding RPL3-containing ribosomes instead of RPL3L-containing ribosomes that are typically found in cardiomyocytes. Using both ribosome profiling (Ribo-Seq) and a novel orthogonal approach consisting of ribosome pulldown coupled to nanopore sequencing (Nano-TRAP), we find that RPL3L neither modulated translational efficiency nor ribosome affinity towards a specific subset of transcripts. By contrast, we show that depletion of RPL3L leads to increased ribosome-mitochondria interactions in cardiomyocytes, which is accompanied by a significant increase in ATP levels, potentially as a result of mitochondrial activity fine-tuning. Our results demonstrate that the existence of tissue-specific RP paralogs does not necessarily lead to enhanced translation of specific transcripts or modulation of translational output. Instead, we reveal a complex cellular scenario in which RPL3L modulates the expression of RPL3, which in turn affects ribosomal subcellular localization and, ultimately, mitochondrial activity.

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Global translational landscape of the Candida albicans morphological transition

G3 Genes|Genome|Genetics ◽

10.1093/g3journal/jkaa043 ◽

2020 ◽

Vol 11 (2) ◽

Author(s):

Vasanthakrishna Mundodi ◽

Saket Choudhary ◽

Andrew D Smith ◽

David Kadosh

Keyword(s):

Candida Albicans ◽

Fungal Pathogens ◽

Global Analysis ◽

Ribosome Profiling ◽

Fine Tuning ◽

Translational Efficiency ◽

Transcriptional Level ◽

Morphological Transition ◽

Wall Functions ◽

Ribosome Pausing

Abstract Candida albicans, a major human fungal pathogen associated with high mortality and/or morbidity rates in a wide variety of immunocompromised individuals, undergoes a reversible morphological transition from yeast to filamentous cells that is required for virulence. While previous studies have identified and characterized global transcriptional mechanisms important for driving this transition, as well as other virulence properties, in C. albicans and other pathogens, considerably little is known about the role of genome-wide translational mechanisms. Using ribosome profiling, we report the first global translational profile associated with C. albicans morphogenesis. Strikingly, many genes involved in pathogenesis, filamentation, and the response to stress show reduced translational efficiency (TE). Several of these genes are known to be strongly induced at the transcriptional level, suggesting that a translational fine-tuning mechanism is in place. We also identify potential upstream open reading frames (uORFs), associated with genes involved in pathogenesis, and novel ORFs, several of which show altered TE during filamentation. Using a novel bioinformatics method for global analysis of ribosome pausing that will be applicable to a wide variety of genetic systems, we demonstrate an enrichment of ribosome pausing sites in C. albicans genes associated with protein synthesis and cell wall functions. Altogether, our results suggest that the C. albicans morphological transition, and most likely additional virulence processes in fungal pathogens, is associated with widespread global alterations in TE that do not simply reflect changes in transcript levels. These alterations affect the expression of many genes associated with processes essential for virulence and pathogenesis.

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Upregulation of 5′-terminal oligopyrimidine mRNA translation upon loss of the ARF tumor suppressor

Scientific Reports ◽

10.1038/s41598-020-79379-8 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Kyle A. Cottrell ◽

Ryan C. Chiou ◽

Jason D. Weber

Keyword(s):

Protein Synthesis ◽

Tumor Cells ◽

Ribosomal Proteins ◽

Human Cancer ◽

Mrna Translation ◽

Ribosome Profiling ◽

Translational Efficiency ◽

Gene Encoding ◽

Terminal Oligopyrimidine ◽

Ribosome Export

AbstractTumor cells require nominal increases in protein synthesis in order to maintain high proliferation rates. As such, tumor cells must acquire enhanced ribosome production. How the numerous mutations in tumor cells ultimately achieve this aberrant production is largely unknown. The gene encoding ARF is the most commonly deleted gene in human cancer. ARF plays a significant role in regulating ribosomal RNA synthesis and processing, ribosome export into the cytoplasm, and global protein synthesis. Utilizing ribosome profiling, we show that ARF is a major suppressor of 5′-terminal oligopyrimidine mRNA translation. Genes with increased translational efficiency following loss of ARF include many ribosomal proteins and translation factors. Knockout of p53 largely phenocopies ARF loss, with increased protein synthesis and expression of 5′-TOP encoded proteins. The 5′-TOP regulators eIF4G1 and LARP1 are upregulated in Arf- and p53-null cells.

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TRIBE editing reveals specific mRNA targets of eIF4E-BP in Drosophila and in mammals

10.1101/2020.02.24.962852 ◽

2020 ◽

Cited By ~ 2

Author(s):

Hua Jin ◽

Daxiang Na ◽

Reazur Rahman ◽

Weijin Xu ◽

Allegra Fieldsend ◽

...

Keyword(s):

Rna Binding ◽

Rna Binding Proteins ◽

Growth Control ◽

Ribosome Profiling ◽

Translational Efficiency ◽

Mtor Inhibition ◽

Mrna Targets ◽

Specific Mrnas ◽

Specific Mrna

Abstract4E-BP (eIF4E-BP) represses translation initiation by binding to the 5’cap-binding protein eIF4E and inhibiting its activity. Although 4E-BP has been shown to be important in growth control, stress response, cancer, neuronal activity and mammalian circadian rhythms, it is not understood how it preferentially represses a subset of mRNAs. We successfully used hyperTRIBE (Targets of RNA-binding proteins identified by editing) to identify in vivo 4E-BP mRNA targets in both Drosophila and mammals under conditions known to activate 4E-BP. The protein associates with specific mRNAs, and ribosome profiling data show that mTOR inhibition changes the translational efficiency of 4E-BP TRIBE targets compared to non-targets. In both systems, these targets have specific motifs and are enriched in translation-related pathways, which correlate well with the known activity of 4E-BP and suggest that it modulates the binding specificity of eIF4E and contributes to mTOR translational specificity.

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ARF suppresses 5’-terminal oligopyrimidine mRNA translation

10.1101/2020.02.07.939165 ◽

2020 ◽

Author(s):

Kyle A. Cottrell ◽

Ryan C. Chiou ◽

Jason D. Weber

Keyword(s):

Protein Synthesis ◽

Tumor Cells ◽

Ribosomal Proteins ◽

Human Cancer ◽

Mrna Translation ◽

Ribosome Profiling ◽

Translational Efficiency ◽

Gene Encoding ◽

Terminal Oligopyrimidine ◽

Ribosome Export

AbstractTumor cells require nominal increases in protein synthesis in order to maintain high proliferation rates. As such, tumor cells must acquire enhanced ribosome production. How many of the mutations in tumor cells ultimately achieve this aberrant production is largely unknown. The gene encoding ARF is the most commonly deleted gene in human cancer. ARF plays a significant role in regulating ribosomal RNA synthesis and processing, ribosome export into the cytoplasm, and global protein synthesis. Utilizing ribosome profiling, we show that ARF is a major suppressor of 5’-terminal oligopyrimidine mRNA translation. Genes with increased translational efficiency following loss of ARF include many ribosomal proteins and translation factors. Knockout of p53 caused a similar increase in 5’-TOP mRNA translation. The 5’-TOP regulators mTORC1, eIF4G1 and LARP1 are dysregulated in ARF and p53 null cells.

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T-Cell Intracellular Antigens and Hu Antigen R Antagonistically Modulate Mitochondrial Activity and Dynamics by Regulating Optic Atrophy 1 Gene Expression

Molecular and Cellular Biology ◽

10.1128/mcb.00174-17 ◽

2017 ◽

Vol 37 (17) ◽

Cited By ~ 10

Author(s):

Isabel Carrascoso ◽

José Alcalde ◽

Carmen Sánchez-Jiménez ◽

Paloma González-Sánchez ◽

José M. Izquierdo

Keyword(s):

T Cell ◽

Optic Atrophy ◽

Morphological Changes ◽

Dynamic Network ◽

Mitochondrial Activity ◽

Translational Efficiency ◽

Mrna Isoforms ◽

Mitochondrial Dynamic ◽

Mitochondrial Dna Damage ◽

Hu Antigen R

ABSTRACT Mitochondria undergo frequent morphological changes to control their function. We show here that T-cell intracellular antigens (TIA1b/TIARb) and Hu antigen R (HuR) have antagonistic roles in mitochondrial function by modulating the expression of mitochondrial shaping proteins. Expression of TIA1b/TIARb alters the mitochondrial dynamic network by enhancing fission and clustering, which is accompanied by a decrease in respiration. In contrast, HuR expression promotes fusion and cristae remodeling and increases respiratory activity. Mechanistically, TIA proteins downregulate the expression of optic atrophy 1 (OPA1) protein via switching of the splicing patterns of OPA1 to facilitate the production of OPA1 variant 5 (OPA1v5). Conversely, HuR enhances the expression of OPA1 mRNA isoforms through increasing steady-state levels and targeting translational efficiency at the 3′ untranslated region. Knockdown of TIA1/TIAR or HuR partially reversed the expression profile of OPA1, whereas knockdown of OPA1 or overexpression of OPA1v5 provoked mitochondrial clustering. Middle-term expression of TIA1b/TIARb triggers reactive oxygen species production and mitochondrial DNA damage, which is accompanied by mitophagy, autophagy, and apoptosis. In contrast, HuR expression promotes mitochondrion-dependent cell proliferation. Collectively, these results provide molecular insights into the antagonistic functions of TIA1b/TIARb and HuR in mitochondrial activity dynamics and suggest that their balance might contribute to mitochondrial physiopathology.

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Ochratoxin A affects oocyte maturation and subsequent embryo developmental dynamics in the juvenile sheep model

Mycotoxin Research ◽

10.1007/s12550-020-00410-y ◽

2020 ◽

Author(s):

Maria Elena Dell’Aquila ◽

Shafaq Asif ◽

Letizia Temerario ◽

Antonella Mastrorocco ◽

Giuseppina Marzano ◽

...

Keyword(s):

Ochratoxin A ◽

Embryo Development ◽

Oocyte Maturation ◽

Oxidative Status ◽

Fine Tuning ◽

Mitochondrial Activity ◽

Sheep Model ◽

High Exposure ◽

Low Levels

Abstract The genotoxic and nephrotoxic mycotoxin Ochratoxin A (OTA) has also been reported to have adverse effects on oocyte maturation and embryo development. Previous studies on the effects of OTA on female fertility have used micromolar concentrations, but no information is available to date on effects in a more relevant nanomolar range. This study used a juvenile sheep model to evaluate the effects of oocyte exposure to low levels of OTA on maturation, fertilization, and embryo development. Further, it was investigated whether different mechanisms of action of OTA could be responsible for varying toxic effects at different levels of exposure. Cumulus-oocyte-complexes (COCs) were exposed to 10 μmol/L–0.1 nmol/L OTA during in vitro maturation and evaluated for cumulus viability, oocyte maturation, and bioenergetic/oxidative status. COCs were subjected to in vitro fertilization, embryo culture, and embryo quality assessment via morphology, viability, bioenergetic/oxidative status, and time-lapse monitoring. At micromolar concentrations, OTA induced cytotoxic effects, by reducing cumulus expansion and oocyte maturation. OTA altered temporospatial dynamics of zygote pronuclear formation and embryo morphokinetics. Blastocysts, even morphologically normal, were found to undergo collapse events, which were probably related to boosted blastocyst mitochondrial activity. At nanomolar concentrations, OTA did not affect COC morpho-functional parameters, but impaired oocyte ability to prevent polyspermy and increased blastocyst apoptosis. In conclusion, in the female germ cell, cytotoxic nonspecific effects characterize OTA-induced toxicity at high exposure levels, whereas fine tuning-mode effects, not associated with altered cell viability and integrity, characterize OTA toxic action at low levels.

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RiboToolkit: an integrated platform for analysis and annotation of ribosome profiling data to decode mRNA translation at codon resolution

Nucleic Acids Research ◽

10.1093/nar/gkaa395 ◽

2020 ◽

Vol 48 (W1) ◽

pp. W218-W229 ◽

Cited By ~ 1

Author(s):

Qi Liu ◽

Tanya Shvarts ◽

Piotr Sliz ◽

Richard I Gregory

Keyword(s):

Mrna Translation ◽

Ribosome Profiling ◽

Open Reading Frames ◽

Translational Efficiency ◽

Translation Efficiency ◽

Sequencing Data ◽

Experimental Conditions ◽

Web Based ◽

Rna Translation ◽

Web Interfaces

Abstract Ribosome profiling (Ribo-seq) is a powerful technology for globally monitoring RNA translation; ranging from codon occupancy profiling, identification of actively translated open reading frames (ORFs), to the quantification of translational efficiency under various physiological or experimental conditions. However, analyzing and decoding translation information from Ribo-seq data is not trivial. Although there are many existing tools to analyze Ribo-seq data, most of these tools are designed for specific or limited functionalities and an easy-to-use integrated tool to analyze Ribo-seq data is lacking. Fortunately, the small size (26–34 nt) of ribosome protected fragments (RPFs) in Ribo-seq and the relatively small amount of sequencing data greatly facilitates the development of such a web platform, which is easy to manipulate for users with or without bioinformatic expertise. Thus, we developed RiboToolkit (http://rnabioinfor.tch.harvard.edu/RiboToolkit), a convenient, freely available, web-based service to centralize Ribo-seq data analyses, including data cleaning and quality evaluation, expression analysis based on RPFs, codon occupancy, translation efficiency analysis, differential translation analysis, functional annotation, translation metagene analysis, and identification of actively translated ORFs. Besides, easy-to-use web interfaces were developed to facilitate data analysis and intuitively visualize results. Thus, RiboToolkit will greatly facilitate the study of mRNA translation based on ribosome profiling.

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DeepShape: estimating isoform-level ribosome abundance and distribution with Ribo-seq data

BMC Bioinformatics ◽

10.1186/s12859-019-3244-0 ◽

2019 ◽

Vol 20 (S24) ◽

Cited By ~ 1

Author(s):

Hongfei Cui ◽

Hailin Hu ◽

Jianyang Zeng ◽

Ting Chen

Keyword(s):

Transcript Level ◽

Transcript Abundance ◽

Ribosome Profiling ◽

Computational Method ◽

Translational Efficiency ◽

Rna Seq ◽

Basic Step ◽

Synonymous Codons ◽

Abundance And Distribution ◽

Different Characteristics

Abstract Background Ribosome profiling brings insight to the process of translation. A basic step in profile construction at transcript level is to map Ribo-seq data to transcripts, and then assign a huge number of multiple-mapped reads to similar isoforms. Existing methods either discard the multiple mapped-reads, or allocate them randomly, or assign them proportionally according to transcript abundance estimated from RNA-seq data. Results Here we present DeepShape, an RNA-seq free computational method to estimate ribosome abundance of isoforms, and simultaneously compute their ribosome profiles using a deep learning model. Our simulation results demonstrate that DeepShape can provide more accurate estimations on both ribosome abundance and profiles when compared to state-of-the-art methods. We applied DeepShape to a set of Ribo-seq data from PC3 human prostate cancer cells with and without PP242 treatment. In the four cell invasion/metastasis genes that are translationally regulated by PP242 treatment, different isoforms show very different characteristics of translational efficiency and regulation patterns. Transcript level ribosome distributions were analyzed by “Codon Residence Index (CRI)” proposed in this study to investigate the relative speed that a ribosome moves on a codon compared to its synonymous codons. We observe consistent CRI patterns in PC3 cells. We found that the translation of several codons could be regulated by PP242 treatment. Conclusion In summary, we demonstrate that DeepShape can serve as a powerful tool for Ribo-seq data analysis.

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Prolonged ovarian storage of mature Drosophila oocytes dramatically increases meiotic spindle instability

eLife ◽

10.7554/elife.49455 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 5

Author(s):

Ethan J Greenblatt ◽

Rebecca Obniski ◽

Claire Mical ◽

Allan C Spradling

Keyword(s):

Meiotic Chromosome ◽

Chromosome Instability ◽

Oocyte Quality ◽

Ribosome Profiling ◽

Meiotic Spindle ◽

Human Embryos ◽

Prolonged Storage ◽

Translational Efficiency ◽

Human Oocytes ◽

In The Wild

Human oocytes frequently generate aneuploid embryos that subsequently miscarry. In contrast, Drosophila oocytes from outbred laboratory stocks develop fully regardless of maternal age. Since mature Drosophila oocytes are not extensively stored in the ovary under laboratory conditions like they are in the wild, we developed a system to investigate how storage affects oocyte quality. The developmental capacity of stored mature Drosophila oocytes decays in a precise manner over 14 days at 25°C. These oocytes are transcriptionally inactive and persist using ongoing translation of stored mRNAs. Ribosome profiling revealed a progressive 2.3-fold decline in average translational efficiency during storage that correlates with oocyte functional decay. Although normal bipolar meiotic spindles predominate during the first week, oocytes stored for longer periods increasingly show tripolar, monopolar and other spindle defects, and give rise to embryos that fail to develop due to aneuploidy. Thus, meiotic chromosome segregation in mature Drosophila oocytes is uniquely sensitive to prolonged storage. Our work suggests the chromosome instability of human embryos could be mitigated by reducing the period of time mature human oocytes are stored in the ovary prior to ovulation.

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RPS15 mutations rewire RNA translation in chronic lymphocytic leukemia

Blood Advances ◽

10.1182/bloodadvances.2020001717 ◽

2021 ◽

Vol 5 (13) ◽

pp. 2788-2792

Author(s):

Stavroula Ntoufa ◽

Marina Gerousi ◽

Stamatia Laidou ◽

Fotis Psomopoulos ◽

Georgios Tsiolas ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Ribosomal Proteins ◽

Ribosomal Subunit ◽

Regulatory Elements ◽

Ribosome Profiling ◽

Lymphocytic Leukemia ◽

Translational Efficiency ◽

Translation Efficiency ◽

Wild Type ◽

Recurrent Mutations

Abstract Recent studies of chronic lymphocytic leukemia (CLL) have reported recurrent mutations in the RPS15 gene, which encodes the ribosomal protein S15 (RPS15), a component of the 40S ribosomal subunit. Despite some evidence about the role of mutant RPS15 (mostly obtained from the analysis of cell lines), the precise impact of RPS15 mutations on the translational program in primary CLL cells remains largely unexplored. Here, using RNA sequencing and ribosome profiling, a technique that involves measuring translational efficiency, we sought to obtain global insight into changes in translation induced by RPS15 mutations in CLL cells. To this end, we evaluated primary CLL cells from patients with wild-type or mutant RPS15 as well as MEC1 CLL cells transfected with mutant or wild-type RPS15. Our data indicate that RPS15 mutations rewire the translation program of primary CLL cells by reducing their translational efficiency, an effect not seen in MEC1 cells. In detail, RPS15 mutant primary CLL cells displayed altered translation efficiency of other ribosomal proteins and regulatory elements that affect key cell processes, such as the translational machinery and immune signaling, as well as genes known to be implicated in CLL, hence highlighting a relevant role for RPS15 in the natural history of CLL.

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