In utero pulse-injection of isotopic amino acids quantifies protein turnover rates during murine fetal development
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive HF-X, Orbitrap Fusion, Q Exactive
ORGANISM(S): Mus Musculus (ncbitaxon:10090)
SUBMITTER: 
Benjamin A. Garcia
PROVIDER: MSV000087342 | MassIVE | Fri Apr 30 07:04:00 BST 2021
SECONDARY ACCESSION(S): PXD025742
REPOSITORIES: MassIVE
Publications
In utero pulse injection of isotopic amino acids quantifies protein turnover rates during murine fetal development.
Baeza Josue J Coons Barbara E BE Lin Zongtao Z Riley John J Mendoza Mariel M Peranteau William H WH Garcia Benjamin A BA
Cell reports methods 20240201 2
Protein translational control is critical for ensuring that the fetus develops correctly and that necessary organs and tissues are formed and functional. We developed an in utero method to quantify tissue-specific protein dynamics by monitoring amino acid incorporation into the proteome after pulse injection. Fetuses of pregnant mice were injected with isotopically labeled lysine and arginine via the vitelline vein at various embyonic days, and organs and tissues were harvested. By analyzing the ...[more]
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Project description:Protein translational control is highly regulated step in the gene expression program during mammalian development that is critical for ensuring that the fetus develops correctly and that all of the necessary organs and tissues are formed and functional. Defects in protein expression during fetal development can lead to severe developmental abnormalities or premature death. Currently, quantitative techniques to monitor protein synthesis rates in a developing fetus (in utero) are limited. Here, we developed a novel in utero stable isotope labeling approach to quantify tissue-specific protein dynamics of the nascent proteome during mouse fetal development. Fetuses of pregnant C57BL/6J mice were injected with isotopically labeled lysine (Lys8) and arginine (Arg10) via the vitelline vein at various gestational days. After treatment, fetal organs/tissues including brain, liver, lung, and heart were harvested for sample preparation and proteomic analysis. We show that the mean incorporation rate for injected amino acids into all organs was 17.50 ± 0.6%. By analyzing the nascent proteome, unique signatures of each tissue were identified by hierarchical clustering. In addition, the quantified proteome-wide turnover rates (kobs) were calculated between 3.81E-5 and 0.424 hour-1. We observed similar protein turnover profiles for analyzed organs (e.g., liver versus brain), however, their distributions of turnover rates vary significantly. The translational kinetic profiles of developing organs displayed differentially expressed protein pathways and synthesis rates which correlated with known physiological changes during mouse development.
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Project description:Functional specification of mammalian tissues is a result of precise regulation of gene expression during development. Although previous transcriptomic and proteomic analyses have provided great biological insight into tissue specific gene expression and their physiological relevance in development, our understanding of translational regulation in developing tissues is lacking. Here, we report a spatio-temporally resolved translatome analysis of six mouse tissues at embryonic and adult stages to quantify the effects of translational regulation and identify new translational components. We quantified the spatial and temporal divergence of gene expression and showed specific changes in gene expression and pathways underlying the divergence. We further showed dynamic translational control by modulating translational efficiency, enhancing tissue specificity during development. We discovered thousands of actively translated upstream open read frames (ORFs) that exhibited spatio-temporal patterns and demonstrated their regulatory roles in translational regulation. Finally, we identified known and novel micropeptides encoded by small ORFs from long non-coding RNAs with functional relevance to tissue development. Our data and analyses facilitate a better understanding of complex translational regulation across tissue and developmental spectra and serve as a useful resource of mouse translatome.
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Project description:Transcriptional silencing of the FMR1 gene in fragile X syndrome (FXS) leads to loss of the RNA-binding protein, FMRP. In addition to its well-established role of regulating protein synthesis, emerging evidence suggests that FMRP acts to coordinate proliferation and differentiation during early neural development. However, whether translational control by FMRP drives critical cellular events of fate specification and developmental transitions in the developing human brain remains unknown. Here, we have designed a high-throughput, in vitro assay that allows for the simultaneous quantification of protein synthesis and proliferation within defined neural subpopulations. Using iPSC-derived neural progenitor cells and organoids to model human cortical neurogenesis in FXS, we show that abnormal protein synthesis during early development alters cellular decisions to favor proliferative over neurogenic cell fates.
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Project description:This model is described in the article:
Kinetics of histone gene expression during early development of Xenopus laevis.
Koster JG, Destrée OH and Westerhoff HV. J Theor Biol.
1988 Nov 21;135(2):139-67. PMID: 3267765
Abstract:
Using literature data for transcriptional and translational rate constants, gene copy numbers, DNA concentrations, and stability constants, we have calculated the expected concentrations of histones and histone mRNA during embryogenesis of Xenopus laevis. The results led us to conclude that: (i) for X. laevis the gene copy number of the histone genes is too low to ensure the synthesis of sufficient histones during very early development, inheritance from the oocyte of either histone protein or histone mRNA (but not necessarily both) is necessary; (ii) from the known storage of histones in the oocyte and the rates of histone synthesis determined by Adamson and Woodland (1977), there would be sufficient histones to structure the newly synthesized DNA up to gastrulation but not thereafter (these empirical rates of histone synthesis may be underestimates); (iii) on the other hand, the amount of H3 mRNA recently observed during early embryogenesis (Koster, 1987, Koster et al., 1988) could direct a higher and sufficient synthesis of H3 protein, also after gastrulation. We present a quantitative model that accounts both for the observed H3 mRNA concentration as a function of time during embryogenesis and for the synthesis of sufficient histones to structure the DNA throughout early embryogenesis. The model suggests that X. laevis exhibits a major (i.e. some 14-fold) reduction in transcription of histone genes approximately 11 hours after fertilization. This reduction could be due to a decrease in the number of transcribed histone genes, a decreased rate constant of transcription with continued transcription of all the histone genes, and/or a reduction in the time during the cell cycle in which histone mRNA synthesis takes place. Alternatively, the histone mRNA stability might decrease approximately 16-fold 11 hours after fertilization.
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To cite BioModels Database, please use: Li C, Donizelli M, Rodriguez N, Dharuri H, Endler L, Chelliah V, Li L, He E, Henry A, Stefan MI, Snoep JL, Hucka M, Le Novère N, Laibe C (2010) BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models. BMC Syst Biol., 4:92.
2005-01-01 | MODEL5954483266 | BioModels
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Project description:RNA-protein interactions mediate a host of cellular processes, underscoring the need for methods to quantify their occurrence in living cells. RNA interaction frequencies for the average cellular protein are undefined, however, and there is no quantitative threshold to define a protein as an RNA-binding protein (RBP). Ultraviolet (UV) cross-linking immunoprecipitation (CLIP)-sequencing, an effective widely used means of characterizing RNA-protein interactions, would particularly benefit from the capacity to quantitate the number of RNA cross-links per protein per cell. In addition, CLIP-seq methods are difficult, have high experimental failure rates and many ambiguous analytical decisions. To address these issues, the easyCLIP method was developed and used to quantify RNA-protein interactions for a panel of known RBPs as well as a spectrum of random non-RBP proteins. easyCLIP provides the advantages of good efficiency compared to current standards, a simple protocol with a very low failure rate, troubleshooting information that includes direct visualization of prepared libraries without amplification, and a new form of analysis. easyCLIP, which uses sequential on-bead ligation of 5’ and 3’ adaptors tagged with different infrared dyes, classified non-RBPs as those with a per protein RNA cross-link rate of <0.1%, with most RBPs substantially above this threshold, including Rbfox1 (18%), hnRNPC (22%), CELF1 (11%), FBL (2%), and STAU1 (1%). easyCLIP with the PCBP1L100 RBP mutant recurrently seen in cancer quantified increased RNA binding compared to wild-type PCBP1 and suggested a potential mechanism for this RBP mutant in cancer. easyCLIP provides a simple, efficient and robust method to both obtain both traditional CLIP-seq information and to define actual RNA interaction frequencies for a given protein, enabling quantitative cross-RBP comparisons as well as insight into RBP mechanisms.
2019-05-17 | GSE131210 | GEO
Project description:Stem cell differentiation is known to involve changes in RNA expression, but little is known about translational control during differentiation. We comprehensively profiled gene expression during differentiation of embryonic stem cells (ESCs) into embyroid bodies (EBs) by integrating conventional transcriptome analysis with global assessment of ribosome loading. Differentiation was accompanied by an anabolic switch, characterized by global increases in transcript abundance, polysome content, protein synthesis rates and protein content. Furthermore, 78% of expressed transcripts showed increased ribosome loading, thereby enhancing translational efficiency. Elevated protein synthesis was accompanied by enhanced phosphorylation of eIF-4E binding protein, suggesting regulation by the mTOR pathway. Some transcripts were under exclusive translational control, including Activated Transcription Factor 5 (ATF5) a b-zip transcription factor, Deleted in Colon Cancer (DCC) the tumor suppressor and Wnt1, the beta-catenin agonist. Parsimonious translation in ESCs may provide a layer of quality control to prevent inappropriate gene expression in the pluripotent state. Keywords: Translation state array analysis during embryonic stem cell differentiation
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Project description:Translational control is a widespread mode of gene regulation in organisms ranging from bacteria to mammals. Computational models posit that translational control of protein expression during elongation is exerted through a traffic jam of multiple ribosomes at ribosome pause sites on mRNAs. Yet neither the in vivo frequency of ribosome traffic jams nor the contribution of such traffic jams to protein expression has been measured in any organism. Here we show that upon starvation for single amino acids in the bacterium Escherichia coli, ribosome traffic jams are pervasive across the transcriptome, but they occur at only a subset of codons cognate to the limiting amino acid, and their severity is determined by the translation efficiency of mRNAs. Surprisingly, a computational model based on the observed traffic jams at ribosome pause sites is quantitatively inconsistent with measured protein synthesis rates. By comparison, a model incorporating abortion of protein synthesis at ribosome pause sites in addition to ribosome traffic jams predicts protein synthesis rate with higher accuracy. Consistent with the latter model, a significant fraction of the nascent polypeptides at ribosome pause sites is degraded through the activity of the transfer-messenger RNA during amino acid starvation in E. coli. Our work provides a minimal, experimentally-constrained model for predicting protein expression from ribosome dynamics, and it suggests the existence of a trade-off between the cellular translational capacity and the processivity of protein synthesis in vivo. 6 samples for ribosome profiling and 5 samples for total mRNA profiling
2014-05-15 | E-GEOD-51052 | biostudies-arrayexpress
Project description:The model encodes the general biological process of protein synthesis and post-translational modification (PTM, such as protein phosphorylation), viewed through the eyes of a specific, widely-used experimental method. Specifically, we model measurements of pulsed stable isotope labelling of amino acids in cell culture (pSILAC), which is a method often used to quantify protein turnover (i.e. the degradation of old and replacement with new) of proteins in a cell.
2024-09-02 | BIOMD0000001078 | BioModels
Project description:eIF4E, the major cap-binding protein, has long been considered limiting for translating the mammalian genome. However, the requirement for eIF4E dose at an organismal level remains unexplored. By generating an Eif4e haploinsufficient mouse, we surprisingly found that 50% reduction in eIF4E, while compatible with normal development and global protein synthesis, significantly impeded cellular transformation and tumorigenesis. Genome-wide translational profiling uncovered a translational program induced by oncogenic transformation and revealed a critical role for eIF4E dose specifically in translating a network of mRNAs enriched for a unique 5’UTR signature. In particular, we demonstrate that eIF4E dose is essential for translating mRNAs regulating reactive oxygen species (ROS) that fuel transformation and cancer cell survival in vivo. Therefore, mammalian cells have evolved surplus eIF4E levels that cancer cells hijack to drive a translational program supporting tumorigenesis Total cellular RNA and high MW polysome associated RNA were isolated from matched untransformed and transformed WT and Eif4e+/- MEFs for analysis on Affymetrix Mouse Gene 1.0 ST arrays. The difference in log2 RMA intensity between matched polysomal RNA and total RNA was taken to quantify translational efficiency (TE).
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