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BONCAT enables time-resolved analysis of protein synthesis in native plant tissue

ABSTRACT: Pulsing the non-canonical amino acid azidohomoalanine into Arabidopsis thaliana seedlings enables in-gel visualization, physical enrichment, and identification of newly synthesized proteins.

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

SUBMITTER: David Tirrell

PROVIDER: PXD005577 | JPOST Repository | Mon Jul 03 00:00:00 BST 2017

REPOSITORIES: jPOST

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Dataset's files

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			Action	DRS
	Wes_HS1.raw	Raw
	Wes_HS2.raw	Raw
	Wes_HS3.raw	Raw
	Wes_Recovery1.raw	Raw
	Wes_Recovery2.raw	Raw

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Publications

Bioorthogonal Noncanonical Amino Acid Tagging (BONCAT) Enables Time-Resolved Analysis of Protein Synthesis in Native Plant Tissue.

Glenn Weslee S WS Stone Shannon E SE Ho Samuel H SH Sweredoski Michael J MJ Moradian Annie A Hess Sonja S Bailey-Serres Julia J Tirrell David A DA

Plant physiology 20170119 3

Proteomic plasticity undergirds stress responses in plants, and understanding such responses requires accurate measurement of the extent to which proteins levels are adjusted to counter external stimuli. Here, we adapt bioorthogonal noncanonical amino acid tagging (BONCAT) to interrogate protein synthesis in vegetative Arabidopsis (<i>Arabidopsis thaliana</i>) seedlings. BONCAT relies on the translational incorporation of a noncanonical amino acid probe into cellular proteins. In this study, the ...[more]

PMID: 28104718

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NASC-seq monitors RNA synthesis in single cells

Project description:Sequencing of newly synthesized RNA can monitor transcriptional dynamics with great sensitivity and high temporal resolution, but is currently restricted to populations of cells. Here, we develop new transcriptome alkylation-dependent single-cell RNA sequencing (NASC-seq), to monitor newly synthesized and pre-existing RNA in single cells. We validate the method on pre-alkylated RNA, and by demonstrating that more newly synthesized RNA was detected for genes with known high mRNA turnover. NASC-seq reveals rapidly up- and down-regulated genes during T-cell activation, and RNA arising from induced genes is essentially only newly synthesized. The newly synthesized and pre-existing transcriptomes after T-cell activation are distinct, confirming that we simultaneously measure gene expression at two time points in single cells. Altogether, NASC-seq is an accessible and powerful tool to investigate transcriptional dynamics that enables the precise monitoring of RNA synthesis at flexible time periods during homeostasis, perturbation responses and cellular differentiation.

2019-07-18 | GSE128273 | GEO

Native molecule sequencing by nano-ID reveals synthesis and stability of RNA isoforms

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Arabidopsis thaliana growth time series

Project description:Transcriptional profiling of A. thaliana seedlings (early development) at 7 time points We investigate biomass heterosis by scoring partial correlations of transcriptional profiles.

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Native Prohibitin Complex Enrichment

Project description:Folding of newly synthesized proteins poses challenges for a functional proteome. Dedicated protein quality control (PQC) systems promote either the folding of nascent polypeptides at ribosomes or, if this fails, ensure their degradation. While well studied for cytosolic protein biogenesis, it is not understood how these processes work for mitochondrially-encoded proteins, key subunits of the oxidative phosphorylation system (OXPHOS). Here, we identify dedicated hubs in proximity to mitoribosomal tunnel exits coordinating mitochondrial translation, protein import, assembly, and protein turnover. Conserved prohibitin/m-AAA protease supercomplexes and the availability of assembly chaperones determine the fate of newly synthesized proteins by molecular triaging. The localization of these competing activities in vicinity to the mitoribosomal tunnel exit allows for a prompt decision whether newly synthesized proteins are fed into OXPHOS assembly or degraded. This repository contains the results of the FLAG-based enrichment of the native prohibitin complex. Please note that we measured elution either with LDS sample buffer or using FLAG-peptides. In the published study, the LDS elution buffer was shown.

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Quantitative Chemical Proteomics Profiling of de novo Proteins Synthesis during Starvation-Mediated Autophagy

Project description:Autophagy is an intracellular degradation mechanism in response to nutrient starvation. Via autophagy, some non-essential cellular constituents are degraded in a lysosome-dependent manner to generate biomolecules that can be utilized for maintaining the metabolic homeostasis. Although it is known that under starvation, the global protein synthesis is significantly reduced mainly due to suppression of mechanistic target of rapamycin (MTOR), there is emerging evidence demonstrating that de novo protein synthesis is involved in the autophagic process. However, characterizing these de novo proteins has been an issue with current techniques. Here, we developed a novel method to identify newly synthesized proteins during starvation-mediated autophagy by combining bio-orthogonal non-canonical amino acid tagging (BONCAT) and isobaric tags for relative and absolute quantitation (iTRAQ). Using bio-orthogonal metabolic tagging, L-azidohomoalanine (AHA) was incorporated into newly synthesized proteins which were then enriched with avidin beads after a click reaction between alkyne bearing biotin and AHA’s bio-orthogonal azide moiety. The enriched proteins were subjected to iTRAQ labeling for protein identification and quantification using liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 711 proteins were identified. The characterized functional profiles of these newly synthesized proteins by bioinformatics analysis suggest their roles in ensuring the pro-survival outcome of autophagy. Finally, we performed validation assays for some selected proteins and found that knockdown of some genes has a significant impact on starvation-induced autophagy and cell death. Thus, the BONCAT-iTRAQ approach is effective in the identification of newly synthesized proteins and provides useful insights to the molecular mechanisms and biological functions of autophagy.

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Transcriptome changes triggered by the synthetic defense elicitors DCA and INA in Arabidopsis thaliana

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An improved, low-cost, hydroponic system for growing Arabidopsis and other model plant species under sterility conditions (Arabidopsis transcriptional profile in early response to Pi starvation)

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Project description:af51_atm - atm time course - which Arabidopsis thaliana genes are induced by ionising radiations? Comparison of SADE transcriptome data (Af1999083) and micro-arrays transcriptome data. - which Arabidopsis thaliana genes are induced by ionising radiations ? time course: 0.75h, 1.5h, 3h, 5h dose: Gamma rays irradiated vs non irradiated 4-days old seedlings Keywords: time course

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Systematic Discovery of Deeply Conserved Plant Protein Complexes, Arabidopsis thaliana, light and dark

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Regulated local synthesis of ribosomal proteins maintains ribosome function in axons

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