scholarly journals Investigation of protein synthesis in Drosophila larvae using puromycin labelling

2017 ◽  
Author(s):  
Lisa Deliu ◽  
Abhishek Ghosh ◽  
Savraj S. Grewal

ABSTRACTTranslational control of gene expression is an important regulator of growth, homeostasis and aging in Drosophila. The ability to measure changes in protein synthesis in response to genetic and environmental cues is therefore important in studying these processes. Here we describe a simple and cost effective approach to assay protein synthesis in Drosophila larval cells and tissues. The method is based on the incorporation of puromycin into nascent peptide chains. Using an ex vivo approach, we label newly synthesized peptides in larvae with puromycin and then measure levels of new protein synthesis using an anti-puromycin antibody. We show that this method can detect changes in protein synthesis in specific cells and tissues in the larvae, either by immunostaining or western blotting. We find that the assay reliably detects changes in protein synthesis induced by two known stimulators of mRNA translation - the nutrient/TORC1 kinase pathway and the transcription factor dMyc. We also use the assay to describe how protein synthesis changes through larval development and in response to two environmental stressors – hypoxia and heat-shock. We propose that this puromycin-labelling assay is a simple but robust method to detect protein synthesis changes at the levels of cells, tissues or whole body in Drosophila.

Development ◽  
2020 ◽  
pp. dev.194480
Author(s):  
Ryo Fujita ◽  
Solène Jamet ◽  
Graham Lean ◽  
Harry Chun Man Cheng ◽  
Steven Hébert ◽  
...  

Translational control of gene expression is an important regulator of adult stem cell quiescence, activation and self-renewal. In skeletal muscle, quiescent satellite cells maintain low levels of protein synthesis, mediated in part through the phosphorylation of eIF2α (P-eIF2α). Pharmacological inhibition of the eIF2α phosphatase with the small molecule sal003 maintains P-eIF2α and permits the expansion of satellite cells ex vivo. Paradoxically, P-eIF2α also increases the translation of specific mRNAs, which is mediated by P-eIF2α dependent readthrough of inhibitory upstream open reading frames (uORFs). Here, we ask whether P-eIF2α dependent mRNA translation enables expansion of satellite cells. Using transcriptomic and proteomic analyses, we show a number of genes associated with the assembly of the spindle pole to be upregulated at the level of protein, without corresponding change in mRNA levels, in satellite cells expanded in the presence of sal003. We show that uORFs in the 5'UTR of mRNA for the mitotic spindle stability gene Tacc3 direct P-eIF2α dependent translation. Satellite cells deficient for TACC3 exhibit defects in expansion, self-renewal and regeneration of skeletal muscle.


2020 ◽  
Author(s):  
Ryo Fujita ◽  
Graham Lean ◽  
Solène Jamet ◽  
Steven Hébert ◽  
Claudia L. Kleinman ◽  
...  

AbstractTranslational control of gene expression is an important regulator of adult stem cell quiescence, activation and self-renewal. In skeletal muscle, quiescent satellite cells maintain low levels of protein synthesis, mediated in part through the phosphorylation of eIF2α (P-eIF2α). Pharmacological inhibition of the eIF2α phosphatase with the small molecule sal003 maintains P-eIF2α and permits the expansion of satellite cells ex vivo. Paradoxically, P-eIF2α also increases the translation of specific mRNAs, which is mediated by P-eIF2α dependent readthrough of inhibitory upstream open reading frames (uORFs). Here, we ask whether P-eIF2α dependent mRNA translation enables expansion of satellite cells. Using transcriptomic and proteomic analyses, we show a number of genes associated with the assembly of the spindle pole to be upregulated at the level of protein, without corresponding change in mRNA levels, in satellite cells expanded in the presence of sal003. We show that uORFs in the 5’UTR of mRNA for the mitotic spindle stability gene Tacc3 direct P-eIF2α dependent translation. Satellite cells deficient for TACC3 exhibit defects in expansion, self-renewal and regeneration of skeletal muscle.SignificanceTranslational control of gene expression has emerged as an important regulator of adult stem cell populations, which maintain low levels of protein synthesis. In adult muscle stem cells, or satellite cells, a portrait of translational control has emerged whereby multiple repression mechanisms prevent the translation of specific mRNAs. It remains unclear how other mRNAs escape repression and are efficiently translated. We show that within the context of low global rates of protein synthesis, satellite cell expansion occurs through the selective translation of Tacc3 mRNA. Tacc3 deficient satellite cells expand poorly, leading to defects in skeletal muscle regeneration. Our study provides a more complete picture of translational control of gene expression in adult stem cell populations.


2001 ◽  
Vol 114 (10) ◽  
pp. 1797-1798
Author(s):  
L.C. Kuhn

Translational Control of Gene Expression edited by N. Sonenberg, J. W. B. Hershey and M. B. Matthews Cold Spring Harbor Laboratory Press (2000) 1020 pages. ISBN 0–87969-568-4 US$115 At the beginning of the 90s most molecular biologists were focusing on transcription and RNA splicing. mRNA translation and its temporal and spatial regulation seemed research topics for insiders at that time. However, all aspects of mRNA fate in the cytoplasm will certainly attract much more attention during the next decade. The field is now flourishing with connections to all disciplines of biology. This book will help you to realize the tremendous variation of translational regulatory mechanisms existing in nature. The evidence for their importance has become so overwhelming that nobody seriously interested in gene expression can ignore it any longer. It is the great merit of the editors of this book that they have brought together an impressive series of first-class reviews written by the most prominent scientists in the field. The new monograph takes a fresh look at the field and is greatly expanded compared with the earlier 1996 version. The book is judiciously divided into two parts. The first part comprises eight broad chapters, giving an overview of the main principles of protein synthesis and its regulation. They serve as a thorough basis for the second part, which comprises twenty-eight chapters, each about 20 pages in length, that present in depth additional exciting areas in which there is strong research activity. Your appetite for this book will be stimulated right at the beginning by the wonderful introductory chapter, which is written jointly by the editors and defines the field in its entire complexity. Given that translation is of course a unifying principle of all living organisms, why are there such a large number of different control mechanisms modulating the use of mRNA templates and making actual protein level not predictable from RNA quantity alone? Are these just remnants of an RNA world or, as the authors seem to believe, effective adaptations for fine-tuning gene expression that have been opportunistically added during evolution? Five broad chapters are devoted to our knowledge of initiation, elongation and termination of translation both in eukaryotes and in prokaryotes. It is amazing how much detail has been added, in just the past five years, to our picture of the biochemistry, structure and function of ribosomes, initiation sites, and translation factors. However, translational control of gene expression is not just a matter of the translation machinery alone. It seems rather that the tremendously versatile mRNA sequences and structures impose the way they are seen by the translation apparatus and its factors. Particularly in eukaryotes, the untranslated parts of mRNAs play a decisive role by providing additional interaction sites for cytoplasmic proteins that modulate mRNA stability, mRNA localization or accessibility of mRNAs to translation. In turn, many of the proteins interacting with mRNA are themselves regulated by metabolites or post-translational modifications. This is beautifully documented in an exciting chapter on the role of translational control in developmental decisions. For example, in Drosophila, a specific cascade of factors acting on RNA localization and translation controls the anterior-posterior body axis. In C. elegans, the fate of germ-line cells is determined by translational repression. And you will find many more such examples. Another important section of the book is devoted to changes in translation that occur during virus infection. Again one is amazed by the variety of ways by which viruses divert the host translation apparatus for their own sake. The shorter chapters give insight into additional exciting areas in the field. For example, research into how heat shock or signal transduction pathways feed into translation, what we know about mRNA degradation of normal and nonsense-containing transcripts, and the evidence that local synaptic protein synthesis represents a molecular hallmark of learning and memory. This book is the most complete and up-to-date review of translational control mechanisms. It is a must for students entering the field, and it will constitute for many years a major reference guide for any investigator who is seriously interested in the full picture of gene expression.


2012 ◽  
Vol 302 (6) ◽  
pp. E674-E686 ◽  
Author(s):  
Samer W. El-Kadi ◽  
Agus Suryawan ◽  
Maria C. Gazzaneo ◽  
Neeraj Srivastava ◽  
Renán A. Orellana ◽  
...  

Orogastric tube feeding is indicated for neonates with impaired ability to ingest and can be administered by intermittent bolus or continuous schedule. Our aim was to determine whether feeding modalities affect muscle protein deposition and to identify mechanisms involved. Neonatal pigs were overnight fasted (FAS) or fed the same amount of food continuously (CON) or intermittently (INT; 7 × 4 h meals) for 29 h. For 8 h, between hours 20 and 28, pigs were infused with [2H5]phenylalanine and [2H2]tyrosine, and amino acid (AA) net balances were measured across the hindquarters. Insulin, branched-chain AA, phenylalanine, and tyrosine arterial concentrations and whole body phenylalanine and tyrosine fluxes were greater for INT after the meal than for CON or FAS. The activation of signaling proteins leading to initiation of mRNA translation, including eukaryotic initiation factor (eIF)4E·eIF4G complex formation in muscle, was enhanced by INT compared with CON feeding or FAS. Signaling proteins of protein degradation were not affected by feeding modalities except for microtubule-associated protein light chain 3-II, which was highest in the FAS. Across the hindquarters, AA net removal increased for INT but not for CON or FAS, with protein deposition greater for INT. This was because protein synthesis increased following feeding for INT but remained unchanged for CON and FAS, whereas there was no change in protein degradation across any dietary treatment. These results suggest that muscle protein accretion in neonates is enhanced with intermittent bolus to a greater extent than continuous feeding, mainly by increased protein synthesis.


2007 ◽  
Vol 35 (6) ◽  
pp. 1634-1637 ◽  
Author(s):  
O.A. Pierrat ◽  
V. Mikitova ◽  
M.S. Bush ◽  
K.S. Browning ◽  
J.H. Doonan

Initiation of mRNA translation is a key regulatory step in the control of gene expression. Microarray analysis indicates that total mRNA levels do not always reflect protein levels, since mRNA association with polyribosomes is necessary for protein synthesis. Phosphorylation of translation initiation factors offers a cost-effective and rapid way to adapt to physiological and environmental changes, and there is increasing evidence that many of these factors are subject to multiple regulatory phosphorylation events. The present article focuses on the nature of reversible phosphorylation and the function of the 5′-cap-binding complex in plants.


2018 ◽  
Author(s):  
Inês Garcez Palha ◽  
Isabelle Anselme ◽  
Sylvie Schneider-Maunoury ◽  
François Giudicelli

ABSTRACTControl of gene expression at the translation level is increasingly regarded as a key feature in many biological processes. Simple, inexpensive, and reliable procedures to visualise sites of protein production are required to allow observation of the spatiotemporal patterns of mRNA translation at subcellular resolution. We present a method, named SPoT (for Subcellular Patterns of Translation), developed upon the original TimeStamp technique (Lin et al., 2008), consisting in the expression of a fluorescent protein fused to a tagged, self-cleavable protease domain. Addition of a cell-permeable protease inhibitor instantly stabilizes newly produced, tagged protein allowing to distinguish recently synthesized protein from more ancient one. After a brief protease inhibitor treatment, the ratio of tagged vs non-tagged forms is highest at sites where proteins are the most recent, i.e. sites of synthesis. Therefore, by comparing tagged and non-tagged protein it is possible to spotlight sites of translation. By specifically expressing the SPoT cassette in neurons of transgenic zebrafish embryos, we reveal sites of neuronal protein synthesis in diverse cellular compartments during early development.


Author(s):  
Gloria J. Wiens ◽  
Anake Umsrithong ◽  
Shawn Miller ◽  
Aneesh Koka ◽  
Travis Vitello

Over the past decade, small satellites have gained the interest of the space industry as a new and cost effective approach for servicing space assets. To address the special constraints inherent to the component miniaturization required for these satellites, researchers in the Space, Automation and Manufacturing Mechanisms Laboratory (SAMM) are exploring foldable mechanisms and their effectiveness for providing autonomous rendezvous and docking capabilities for small space vehicles. This paper focuses particularly on the design of autonomous docking mechanisms for space vehicles within the small satellite class known as picosatellite (size and mass requirements: 1 kilogram mass within a 10×10×10 centimeter cube). The docking mechanisms deployment scenario is a dual satellite system comprised of two small satellites (a chaser and a target). The chaser has attitude and translational control capability, while the target is a passive satellite having only attitude stabilization capability. This paper will first present a review of the existing docking mechanism technology utilized in space. This is followed by details of a foldable mechanism approach for providing small satellites autonomous docking capabilities. This includes geometric and dynamic analysis conducted in ADAMS software simulations.


2018 ◽  
Vol 46 (4) ◽  
pp. 855-869 ◽  
Author(s):  
Huili Guo

The control of translation is increasingly recognized as a major factor in determining protein levels in the cell. The ribosome — the cellular machine that mediates protein synthesis — is typically seen as a key, but invariant, player in this process. This is because translational control is thought to be mediated by other auxiliary factors while ribosome recruitment is seen as the end-point of regulation. However, recent developments have made it clear that heterogeneous ribosome types can exist in different tissues, and more importantly, that these ribosomes can preferentially translate different subsets of mRNAs. In so doing, heterogeneous ribosomes could be key regulatory players in differentiation and development. Here, we examine current evidence for the existence of different ribosome types and how they might arise. In particular, we will take a close look at the mechanisms through which these ribosomes might mediate selective mRNA translation. We also summarize recently developed techniques/approaches that will aid in our understanding of the functions of such specialized ribosomes.


2021 ◽  
Author(s):  
Carla Layana ◽  
Emiliano S. Vilardo ◽  
Gonzalo Corujo ◽  
Greco Hernandez ◽  
Rolando Rivera-Pomar

Eukaryotic translation initiation factor 4E (eIF4E) is a key factor involved in different aspects of mRNA metabolism. Drosophila melanogaster genome encodes eight eIF4E isoforms, and the canonical isoform eIF4E-1 is a ubiquitous protein that plays a key role in mRNA translation. eIF4E-3 is specifically expressed in testis and controls translation during spermatogenesis. In eukaryotic cells, translational control and mRNA decay is highly regulated in different cytoplasmic ribonucleoprotein foci, which include the processing bodies (PBs). In this study, we show that Drosophila eIF4E-1 and eIF4E-3 occur in PBs where might play a role in mRNA storage and translational repression. We also demonstrate that the DEAD-box RNA helicase Me31B, a component of PBs, physically interacts with eIF4E-1 and eIF4E-3 both in the yeast two-hybrid system and FRET in Drosophila S2 cells. Moreover, truncated and point mutated Me31B proteins indicate that the binding sites of Me31B for eIF4E-1 and eIF4E-3 are located in different domains. Residues Y401-L407 (at the carboxy-terminal) are essential for interaction with eIF4E-1, whereas residues F63-L70 (at the amino-terminal) are critical for interaction with eIF4E-3. Thus, Me31B represents a novel type of eIF4E-interacting protein. Our observations suggest that Me31B might recognize different eIF4E isoforms in different tissues, which could be the key to silencing specific messengers. They provide further evidence that alternative forms of eIF4E and their interactions with various partners add complexity to the control of gene expression in eukaryotes.


2002 ◽  
Vol 283 (6) ◽  
pp. E1144-E1150 ◽  
Author(s):  
Stephen J. Crozier ◽  
Douglas R. Bolster ◽  
Ali K. Reiter ◽  
Scot R. Kimball ◽  
Leonard S. Jefferson

The study described herein investigated the role of free fatty acids (FFAs) in the maintenance of protein synthesis in vivo in rat cardiac and skeletal muscle. Suppression of FFA β-oxidation by methyl palmoxirate caused a marked reduction in protein synthesis in the heart. The effect on protein synthesis was mediated in part by changes in the function of eukaryotic initiation factors (eIFs) involved in the initiation of mRNA translation. The guanine nucleotide exchange activity of eIF2B was repressed, phosphorylation of the α-subunit of eIF2 was enhanced, and phosphorylation of eIF4E-binding protein-1 and ribosomal protein S6 kinase was reduced. Similar changes in protein synthesis and translation initiation were not observed in the gastrocnemius following treatment with methyl palmoxirate. In heart, repressed β-oxidation of FFA correlated, as demarcated by changes in the ATP/AMP ratio and phosphorylation of AMP-activated kinase, with alterations in the energy status of the tissue. Therefore, the activation state of signal transduction pathways that are responsive to cellular energy stress represents one mechanism whereby translation initiation may be regulated in cardiac muscle.


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