scholarly journals Accurate design of translational output by a neural network model of ribosome distribution

2017 ◽  
Author(s):  
Robert J Tunney ◽  
Nicholas J McGlincy ◽  
Monica E Graham ◽  
Nicki Naddaf ◽  
Lior Pachter ◽  
...  
Keyword(s):  
Neural Network ◽  
Fluorescent Protein ◽  
Synonymous Codon ◽  
Full Range ◽  
Rna Stability ◽  
Ribosome Profiling ◽  
Translation Elongation ◽  
Accurate Design ◽  
The Impact

Synonymous codon choice can have dramatic effects on ribosome speed, RNA stability, and protein expression. Ribosome profiling experiments have underscored that ribosomes do not move uniformly along mRNAs, exposing a need for models of coding sequences that capture the full range of empirically observed variation. We present a method, Ixnos, that models this variation in translation elongation using a feedforward neural network to predict the translation elongation rate at each codon as a function of its sequence neighborhood. Our approach revealed sequence features affecting translation elongation and quantified the impact of large technical biases in ribosome profiling. We applied our model to design synonymous variants of a fluorescent protein spanning the range of possible translation speeds predicted with our model. We found that levels of the fluorescent protein in yeast closely tracked the predicted translation speeds across their full range. We therefore demonstrate that our model captures information determining translation dynamics in vivo, and that control of translation elongation alone is sufficient to produce large, quantitative differences in protein output.

scholarly journals Phosphorylation within Intrinsic Disordered Region Discriminates Histone Variant macroH2A1 Splicing Isoforms—macroH2A1.1 and macroH2A1.2

Biology ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 659
Author(s):  
Sebastiano Giallongo ◽  
Oriana Lo Re ◽  
Gabriela Lochmanová ◽  
Luca Parca ◽  
Francesco Petrizzelli ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Regulatory Element ◽  
Histone Variants ◽  
Histone H2a ◽  
Human Hepatoma Cells ◽  
Nucleosome Core ◽  
Intrinsically Disordered ◽  
Splicing Isoforms ◽  
The Impact

Background: Gene expression in eukaryotic cells can be governed by histone variants, which replace replication-coupled histones, conferring unique chromatin properties. MacroH2A1 is a histone H2A variant containing a domain highly similar to H2A and a large non-histone (macro) domain. MacroH2A1, in turn, is present in two alternatively exon-spliced isoforms: macroH2A1.1 and macroH2A1.2, which regulate cell plasticity and proliferation in a remarkably distinct manner. The N-terminal and the C-terminal tails of H2A histones stem from the nucleosome core structure and can be target sites for several post-translational modifications (PTMs). MacroH2A1.1 and macroH2A1.2 isoforms differ only in a few amino acids and their ability to bind NAD-derived metabolites, a property allegedly conferring their different functions in vivo. Some of the modifications on the macroH2A1 variant have been identified, such as phosphorylation (T129, S138) and methylation (K18, K123, K239). However, no study to our knowledge has analyzed extensively, and in parallel, the PTM pattern of macroH2A1.1 and macroH2A1.2 in the same experimental setting, which could facilitate the understanding of their distinct biological functions in health and disease. Methods: We used a mass spectrometry-based approach to identify the sites for phosphorylation, acetylation, and methylation in green fluorescent protein (GFP)-tagged macroH2A1.1 and macroH2A1.2 expressed in human hepatoma cells. The impact of selected PTMs on macroH2A1.1 and macroH2A1.2 structure and function are demonstrated using computational analyses. Results: We identified K7 as a new acetylation site in both macroH2A1 isoforms. Quantitative comparison of histone marks between the two isoforms revealed significant differences in the levels of phosphorylated T129 and S170. Our computational analysis provided evidence that the phosphorylation status in the intrinsically disordered linker region in macroH2A1 isoforms might represent a key regulatory element contributing to their distinct biological responses. Conclusions: Taken together, our results report different PTMs on the two macroH2A1 splicing isoforms as responsible for their distinct features and distribution in the cell.

scholarly journals Knockout of a labeled strain of Escherichia coli in the mouse gut using native phages

2020 ◽  
Author(s):  
Li Ping ◽  
Chen Jingchao ◽  
Zhiyu Zhang ◽  
Li Yi ◽  
Liu Lei ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Host Specificity ◽  
Fluorescent Protein ◽  
Bacterial Species ◽  
Sequencing Analysis ◽  
In Vitro Test ◽  
The Impact ◽  
Phage Treatment

Abstract Background: There is a lack of methodological investigation of the in situ functions of a bacterial species in microecosystems such as the animal gut, although the microbiome has become a focus in the microbiology field worldwide. Results: We used native mixed phages containing Escherichia phages T1 and T4 as a microbial editing tool for eliminating Escherichia coli MG1655 labeled with green fluorescent protein in the mouse gut. The phages possessed rigorous host specificity at both the genus and species levels, resulting in an 8.8-log10 decrease in the titer of viable bacteria after 12 h of phage treatment in an in vitro test. In vivo, they knocked out strain MG1655 not only at concentrations of 10 6 -10 8 CFU g -1 colonizing the mouse gut but also even in mice fed with feedstuff containing the bacterium. In addition, the impact of phage treatment on the microbial community structure of the mouse gut was not significantly ( p ＞0.05) based on a 16S rRNA amplicon gene sequencing analysis, although the richness of some bacteria changed significantly. Conclusions: We provide a feasible microbial editing technique for the animal gut. Native phages with strict host specificity can effectively knock out a target bacterium by single or continuous gastric perfusion, with limited perturbation of microbial diversity, which is beneficial for studies of the function of a specific bacterial species colonizing a complicated microecosystem.

scholarly journals Eukaryotic Lipid Body Proteins in Oleogenous Actinomycetes and Their Targeting to Intracellular Triacylglycerol Inclusions: Impact on Models of Lipid Body Biogenesis

2006 ◽  
Vol 72 (10) ◽  
pp. 6743-6750 ◽  
Author(s):  
Jan Hänisch ◽  
Marc Wältermann ◽  
Horst Robenek ◽  
Alexander Steinbüchel
Keyword(s):  
Fluorescent Protein ◽  
Current Model ◽  
Freeze Fracture ◽  
Lipid Body ◽  
Rhodococcus Opacus ◽  
Distribution Analysis ◽  
Lipid Bodies ◽  
Lipid Inclusions ◽  
The Impact

ABSTRACT Bacterial neutral lipid inclusions are structurally related to eukaryotic lipid bodies. These lipid inclusions are composed of a matrix of triacylglycerols (TAGs) or wax esters surrounded by a monolayer of phospholipids. Whereas the monolayers of lipid bodies from animal and plant cells harbor specific classes of proteins which are involved in the structure of the inclusions and lipid homoestasis, no such proteins are known to be associated with bacterial lipid inclusions. The present study was undertaken to reveal whether the mammalian lipid body proteins perilipin A, adipose differentiation-related protein, and tail-interacting protein of 47 kDa (TIP47), which comprise the so called PAT family proteins, and the maize (Zea mays L.) oleosin are targeted to prokaryotic TAG bodies in vivo. When fused to enhanced green fluorescent protein, all proteins except the oleosin were mainly located at the surfaces of lipid inclusions when heterologously expressed in the recombinant actinomycetes Rhodococcus opacus PD630 and Mycobacterium smegmatis mc2155. A more detailed intracellular distribution analysis of TIP47 in recombinant R. opacus cells by immunocytochemical labeling of ultrathin cryosections and freeze fracture replicas revealed a substantial amount of TIP47 protein also pervading the cores of the inclusions. We discuss the impact of these results on the current model of lipid body biogenesis in prokaryotes.

scholarly journals The METTL3/MALAT1/PTBP1/USP8/TAK1 axis promotes pyroptosis and M1 polarization of macrophages and contributes to liver fibrosis

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Bo Shu ◽  
Ying-Xia Zhou ◽  
Hao Li ◽  
Rui-Zhi Zhang ◽  
Chao He ◽  
...  
Keyword(s):  
Liver Fibrosis ◽  
Western Blot ◽  
Rna Stability ◽  
Interferon Γ ◽  
In Vivo Model ◽  
Loss Of Function ◽  
M1 Polarization ◽  
The Impact

AbstractPro-inflammatory M1 macrophages, via activating hepatic stellate cells, contribute to liver fibrosis. In this study, we examined the mechanism and the significance of a signaling axis, METTL3/MALAT1/PTBP1/USP8/TAK1, in regulating pyroptosis and M1 polarization of hepatic macrophages. Liver fibrosis model was established in vivo by CCl4 treatment; M1 polarization was induced in vitro by treating macrophages with lipopolysaccharide or interferon γ. Expressions of METTL3, MALAT1, PTBP1, USP8, and TAK1 were measured by RT-PCR and/or Western blot in Kupffer cells (KCs) isolated from in vivo model or in vitro activated macrophages. Macrophage phenotypes including inflammation (RT-qPCR analysis of a panel of proinflammatory cytokines and ELISA on productions of interleukin (IL)−1β and IL-18) and pyroptosis (Western blot of NLRP3, Caspase-1, and GSDMD) were investigated. The impact of METTL3 on m6A methylation of MALAT1 was examined by methylated RNA immunoprecipitation (RIP), the interaction between PTBP1 and MALAT1 or USP8 mRNA by combining RNA pull-down, RIP, and RNA stability assays, and the crosstalk between USP8 and TAK1 by co-immunoprecipitation and protein degradation assays. Functional significance of individual component of METTL3/MALAT1/PTBP1/USP8/TAK1 axis was assessed by combining gain-of-function and loss-of-function approaches. In KCs isolated from in vivo liver fibrosis model or in vitro M1-polarized macrophages, METTL3 was up-regulated, and sequentially, it increased MALAT1 level via m6A methylation, which promoted USP8 mRNA degradation through the interaction with PTBP1. Reduced USP8 expression regulated the ubiquitination and protein stability of TAK1, which promoted pyroptosis and inflammation of macrophages. The signaling cascade METTL3/MALAT1/PTBP1/USP8/TAK1, by essentially stimulating pyroptosis and inflammation of macrophages, aggravates liver fibrosis. Therefore, targeting individual components of this axis may benefit the treatment of liver fibrosis.

scholarly journals Influence of dietary soy isoflavones on immature hamster uterotrophic and Hershberger assays

2013 ◽  
Vol 57 (4) ◽  
pp. 579-585 ◽  
Author(s):  
Maria Minta ◽  
Lidia Radko ◽  
Sylwia Stypuła-Trębas ◽  
Barbara Woźniak ◽  
Jan Żmudzki
Keyword(s):  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Testosterone Propionate ◽  
Levator Ani ◽  
Ventral Prostate ◽  
Protein Assay ◽  
Green Fluorescent ◽  
High Degree ◽  
The Impact

Abstract To select appropriate diet for hamsters used in the uterotrophic and Hershberger assays two rodent diets were compared: Murigran (Agropol, Poland) and Altromin 7010 (Altromin Spezialfutter GmbH&Co., Germany). The contents of bioactive compounds in feeds were evaluated by liquid chromatography, and their oestrogenic activity by yeast enhanced green fluorescent protein assay. In opposition to Altromin, Murigran contained high amounts (μg/kg) of genistein (765 600) and daidzein (132 000), and the oestrogenic activity of these compounds, expressed as 17β-oestradiol equivalent concentration (EEQ), was found to be 9.54 μg EEQ/kg. In in vivo study, Murigran induced a high degree of oestrogenisation in immature hamsters, and females failed to exhibit a normal uterine response to recommended dose of a model oestrogen agonist 17α-ethinyloestradiol. There was no influence of the diet on the weight of five accessory sex organs (ASO): ventral prostate, seminal vesicles with coagulating glands, levator ani bulbocavernosus muscles, Cowper`s glands, and glans penis of control males. However, the impact on ASO response to model androgen agonist, testosterone propionate was observed. The obtained results provide the evidence that phytooestrogen-rich feed modulates the oestrogenic and androgenic response to chemicals.

scholarly journals Unique actions of GABA arising from cytoplasmic chloride microdomains

2020 ◽  
Author(s):  
Negah Rahmati ◽  
Kieran P. Normoyle ◽  
Joseph Glykys ◽  
Volodymyr I. Dzhala ◽  
Kyle P. Lillis ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Reversal Potential ◽  
Receptor Activation ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging ◽  
Electrophysiological Measurements ◽  
Highly Correlated ◽  
Cation Chloride Cotransporters ◽  
The Impact

AbstractDevelopmental, cellular, and subcellular variations in the direction of neuronal Cl− currents elicited by GABAA receptor activation have been frequently reported, and we found a corresponding variance in the reversal potential (EGABA) for individual interneurons synapsing on a single pyramidal cell. These findings suggest a corresponding variance in the cytoplasmic concentration of Cl− ([Cl−i]). We determined [Cl−]i by: 1) two-photon imaging of the Cl− sensitive, ratiometric fluorescent protein SuperClomeleon (sCLM); 2) Fluorescence Lifetime IMaging (FLIM) of the Cl− sensitive fluorophore MEQ; and 3) electrophysiological measurements of EGABA. These methods collectively demonstrated stable [Cl−]i microdomains in individual neurons in vivo. Fluorometric and electrophysiological estimates of local [Cl−]i were highly correlated. [Cl−]i microdomains persisted after pharmacological inhibition of cation-chloride cotransporters (CCCs) but steadily decreased after inhibiting the polymerization of the anionic macromolecule actin. These studies highlight the existence of functionally significant neuronal Cl− microdomains that modify the impact of GABAergic inputs.

scholarly journals Cell-type–specific, multicolor labeling of endogenous proteins with split fluorescent protein tags in Drosophila

2021 ◽  
Vol 118 (23) ◽  
pp. e2024690118
Author(s):  
Rie Kamiyama ◽  
Kota Banzai ◽  
Peiwei Liu ◽  
Abhijit Marar ◽  
Ryo Tamura ◽  
...  
Keyword(s):  
Tandem Repeats ◽  
Fluorescent Protein ◽  
Protein Localization ◽  
Fluorescence Signal ◽  
Cell Type ◽  
Genomic Locus ◽  
Cell Type Specific ◽  
Endogenous Proteins ◽  
The Impact

The impact of the Drosophila experimental system on studies of modern biology cannot be understated. The ability to tag endogenously expressed proteins is essential to maximize the use of this model organism. Here, we describe a method for labeling endogenous proteins with self-complementing split fluorescent proteins (split FPs) in a cell-type–specific manner in Drosophila. A short fragment of an FP coding sequence is inserted into a specific genomic locus while the remainder of the FP is expressed using an available GAL4 driver line. In consequence, complementation fluorescence allows examination of protein localization in particular cells. Besides, when inserting tandem repeats of the short FP fragment at the same genomic locus, we can substantially enhance the fluorescence signal. The enhanced signal is of great value in live-cell imaging at the subcellular level. We can also accomplish a multicolor labeling system with orthogonal split FPs. However, other orthogonal split FPs do not function for in vivo imaging besides split GFP. Through protein engineering and in vivo functional studies, we report a red split FP that we can use for duplexed visualization of endogenous proteins in intricate Drosophila tissues. Using the two orthogonal split FP systems, we have simultaneously imaged proteins that reside in distinct subsynaptic compartments. Our approach allows us to study the proximity between and localization of multiple proteins endogenously expressed in essentially any cell type in Drosophila.

scholarly journals Monitoring Protein Dynamics in Protein O-Mannosyltransferase Mutants In Vivo by Tandem Fluorescent Protein Timers

Molecules ◽  
2018 ◽  
Vol 23 (10) ◽  
pp. 2622 ◽  
Author(s):  
Joan Castells-Ballester ◽  
Ewa Zatorska ◽  
Matthias Meurer ◽  
Patrick Neubert ◽  
Anke Metschies ◽  
...  
Keyword(s):  
Protein Dynamics ◽  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Model Organism ◽  
Protein Glycosylation ◽  
Major Protein ◽  
Specific Substrate ◽  
Evolutionarily Conserved ◽  
The Impact

For proteins entering the secretory pathway, a major factor contributing to maturation and homeostasis is glycosylation. One relevant type of protein glycosylation is O-mannosylation, which is essential and evolutionarily-conserved in fungi, animals, and humans. Our recent proteome-wide study in the eukaryotic model organism Saccharomyces cerevisiae revealed that more than 26% of all proteins entering the secretory pathway receive O-mannosyl glycans. In a first attempt to understand the impact of O-mannosylation on these proteins, we took advantage of a tandem fluorescent timer (tFT) reporter to monitor different aspects of protein dynamics. We analyzed tFT-reporter fusions of 137 unique O-mannosylated proteins, mainly of the secretory pathway and the plasma membrane, in mutants lacking the major protein O-mannosyltransferases Pmt1, Pmt2, or Pmt4. In these three pmtΔ mutants, a total of 39 individual proteins were clearly affected, and Pmt-specific substrate proteins could be identified. We observed that O-mannosylation may cause both enhanced and diminished protein abundance and/or stability when compromised, and verified our findings on the examples of Axl2-tFT and Kre6-tFT fusion proteins. The identified target proteins are a valuable resource towards unraveling the multiple functions of O-mannosylation at the molecular level.

scholarly journals Transcriptome-wide sites of collided ribosomes reveal principles of translational pausing

2019 ◽  
Author(s):  
Alaaddin Bulak Arpat ◽  
Angélica Liechti ◽  
Mara De Matos ◽  
René Dreos ◽  
Peggy Janich ◽  
...  
Keyword(s):  
Protein Production ◽  
Evolutionary Conservation ◽  
Ribosome Profiling ◽  
Translation Rate ◽  
Translation Elongation ◽  
Peptide Motifs ◽  
Functional Relevance ◽  
Polypeptide Structure ◽  
The Impact ◽  
Pause Site

AbstractTranslation initiation is the major regulatory step defining the rate of protein production from an mRNA. Meanwhile, the impact of non-uniform ribosomal elongation rates is largely unknown. Using a modified ribosome profiling protocol based on footprints from two closely packed ribosomes (disomes), we have mapped ribosomal collisions transcriptome-wide in mouse liver. We uncover that the stacking of an elongating onto a paused ribosome occurs frequently and scales with translation rate, trapping ∼10% of translating ribosomes in the disome state. A distinct class of pause sites, independent of translation rate, is indicative of deterministic pausing signals. Pause site association with specific amino acids, peptide motifs and nascent polypeptide structure, is suggestive of programmed pausing as a widespread mechanism associated with protein folding. Evolutionary conservation at disome sites indicates functional relevance of translational pausing. Collectively, our disome profiling approach allows unique insights into gene regulation occurring at the step of translation elongation.

scholarly journals Multi-resBind: a residual network-based multi-label classifier for in vivo RNA binding prediction and preference visualization

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Shitao Zhao ◽  
Michiaki Hamada
Keyword(s):  
Neural Network ◽  
Deep Learning ◽  
Binding Sites ◽  
Prediction Accuracy ◽  
Large Scale ◽  
Rna Binding ◽  
Binding Prediction ◽  
Binding Preference ◽  
The Impact

Abstract Background Protein-RNA interactions play key roles in many processes regulating gene expression. To understand the underlying binding preference, ultraviolet cross-linking and immunoprecipitation (CLIP)-based methods have been used to identify the binding sites for hundreds of RNA-binding proteins (RBPs) in vivo. Using these large-scale experimental data to infer RNA binding preference and predict missing binding sites has become a great challenge. Some existing deep-learning models have demonstrated high prediction accuracy for individual RBPs. However, it remains difficult to avoid significant bias due to the experimental protocol. The DeepRiPe method was recently developed to solve this problem via introducing multi-task or multi-label learning into this field. However, this method has not reached an ideal level of prediction power due to the weak neural network architecture. Results Compared to the DeepRiPe approach, our Multi-resBind method demonstrated substantial improvements using the same large-scale PAR-CLIP dataset with respect to an increase in the area under the receiver operating characteristic curve and average precision. We conducted extensive experiments to evaluate the impact of various types of input data on the final prediction accuracy. The same approach was used to evaluate the effect of loss functions. Finally, a modified integrated gradient was employed to generate attribution maps. The patterns disentangled from relative contributions according to context offer biological insights into the underlying mechanism of protein-RNA interactions. Conclusions Here, we propose Multi-resBind as a new multi-label deep-learning approach to infer protein-RNA binding preferences and predict novel interactions. The results clearly demonstrate that Multi-resBind is a promising tool to predict unknown binding sites in vivo and gain biology insights into why the neural network makes a given prediction.

Sign in / Sign up

Export Citation Format

Share Document