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Membrane-associated transcripts in Arabidopsis; their isolation...

ABSTRACT: Membrane-associated, integral membrane and secreted proteins are of key importance in many cellular processes. For most of the 28 952 predicted proteins in Arabidopsis, the actual subcellular localisation has not been demonstrated experimentally. So far, their potential membrane-association has been deduced from algorithms that predict transmembrane domains and signal peptides. However, the comprehensiveness and accuracy of these algorithms is still limited. The majority of membrane-associated and secreted proteins is synthesised on membrane-bound polysomes. Therefore, the isolation and characterisation of mRNA associated with membrane-bound polysomes offers an experimental tool for the genome-wide identification of these proteins. Here we describe an efficient method to isolate mRNA from membrane-bound polysomes and report on the validation of the method to enrich for transcripts encoding membrane-associated and secreted proteins. The sensitivity and reproducibility of the isolation method was investigated by DNA microarray analysis. Pearson correlations between transcript levels obtained from three replicate isolations showed that the method is highly reproducible. A significant enrichment for mRNAs encoding proteins containing predicted transmembrane domains and signal peptides was observed in the membrane-bound polysomal fraction. In this fraction, 301 transcripts were classified by gene ontologies as ‘cellular component unknown’, and potentially encode previously unrecognised secreted or membrane-associated proteins. Keywords: mRNA localisation, mRNA fractionation, predict protein localisation

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE4023 | GEO | 2006/03/17

SECONDARY ACCESSION(S): PRJNA95125

REPOSITORIES: GEO

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Membrane-associated transcripts in Arabidopsis; their isolation...

Project description:Membrane-associated, integral membrane and secreted proteins are of key importance in many cellular processes. For most of the 28 952 predicted proteins in Arabidopsis, the actual subcellular localisation has not been demonstrated experimentally. So far, their potential membrane-association has been deduced from algorithms that predict transmembrane domains and signal peptides. However, the comprehensiveness and accuracy of these algorithms is still limited. The majority of membrane-associated and secreted proteins is synthesised on membrane-bound polysomes. Therefore, the isolation and characterisation of mRNA associated with membrane-bound polysomes offers an experimental tool for the genome-wide identification of these proteins. Here we describe an efficient method to isolate mRNA from membrane-bound polysomes and report on the validation of the method to enrich for transcripts encoding membrane-associated and secreted proteins. The sensitivity and reproducibility of the isolation method was investigated by DNA microarray analysis. Pearson correlations between transcript levels obtained from three replicate isolations showed that the method is highly reproducible. A significant enrichment for mRNAs encoding proteins containing predicted transmembrane domains and signal peptides was observed in the membrane-bound polysomal fraction. In this fraction, 301 transcripts were classified by gene ontologies as âcellular component unknownâ, and potentially encode previously unrecognised secreted or membrane-associated proteins. Membrane-bound (MBP) and free polysomes (FP) were isolated in triplicate from one batch of two weeks old aboveground parts of Arabidopsis seedlings. Each combination of isolated MBP and FP was applied to 4 microarrays, of which two were dyeswapped, giving a total of 12 arrays.

2010-07-01 | E-GEOD-4023 | biostudies-arrayexpress

Yeast Membrane Bound Polysome

Project description:Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc. mRNA species bound to membrane-associated polysomes were separated from other mRNAs by sedimentation equilibrium or sedimentation velocity. The distribution of individual transcripts in the 'membrane-bound' and 'cytosolic' fractions was quantitated for thousands of genes by hybridization to DNA microarrays. Transcripts known to encode secreted or membrane proteins were enriched in the membrane-bound fractions, whereas those known to encode cytoplasmic proteins were enriched in the fractions containing mRNAs associated with free and cytoplasmic ribosomes. On this basis, we identified over 275 human genes and 285 yeast genes that are likely to encode previously unrecognized secreted or membrane proteins. Computed

2005-08-24 | E-GEOD-3180 | biostudies-arrayexpress

Meta-organization of Translation Centers Revealed by Proximity Mapping of Endoplasmic Reticulum Ribosome Interactors

Project description:We report a method to use BioID proximity labeling to map the sub-organization of translating mRNAs on the endoplasmic reticulum. This method first isolates membrane bound polysomes using a Sephacryl 400 resin-based enrichment followed by isolation of biotin labeled polysomes by streptavidin beads and high-throughput sequencing of the bound polysomes. We find that, for the two pools of polysomes labeled by either LRRC59 or Sec61beta BioID reporters, there is an interesting divergence in specific mRNAs that are entering into the secretory pathway such that secreted proteins are much more enriched towards the LRRC59 reporter with Sec61beta serving a more varied role in mRNA translation.

2019-04-18 | GSE118873 | GEO

Yeast Membrane Bound Polysome

2005-08-24 | GSE3180 | GEO

AKAP12 UV RNA immunoprecipitation followed by high throughput sequencing

Project description:Regulation of subcellular mRNA localisation is a fundamental biological mechanism, which adds a spatial dimension to the diverse layers of post-transcriptional control of gene expression. Insights into this phenomenon have been described in a multitude of cell types and across taxonomic kingdoms, highlighting its biological significance. The cellular compartment in which mRNAs are located may define distinct aspects of the encoded proteins, ranging from production rate and complex formation to localised activity. Ultimately, mRNA localisation supports compartmentalised mechanistic outputs that can respond to local stimuli, orient migration, or even shape cells. We used an unbiased method to profile the RNA-bound proteome in migrating endothelial cells and found that the plasma membrane (PM)-associated A-kinase anchor protein 12 (AKAP12) interacts with various mRNAs, including mRNAs encoding kinases with Actin remodelling activity. To identify transcripts bound by AKAP12, we carried out UV crosslinking experiments followed by RNA immunoprecipitation and high throughput sequencing. Our data offer novel insights in complex mechanisms of spatial control of gene expression.

2024-03-28 | GSE247682 | GEO

MCF-7 Fractionated RNA

Project description:RNA from MCF-7 cells was fractionated by sucrose density gradient centrifugation to separate RNA associated with membrane-bound polysomes from RNA associated with free polysomes. These two populations were hybridized in triplicate to U133A microarrays. Keywords: repeat sample

2004-12-01 | GSE1400 | GEO

Transcription profiling of membrane-bound and cytosolic polysomes isolated from human MCF-7 cells

2007-07-29 | E-GEOD-1400 | biostudies-arrayexpress

Exhaustive profiling in Arabidopsis reveals abundant polysome-associated 24-nt small RNAs including hitherto undescribed AGO5-associated pseudogene-derived siRNAs (psiRNAs) [RNA]

Project description:The present study was primarily aimed at expanding the scope of the previous analyses, which focused largely on AGO1-miRNA interactions with total polysomes and membrane-bound polysomes (MBPs), respectively. Our larger aim was to comprehensively analyze which, among the multiple AGO effector proteins and the numerous cellular sRNA classes of Arabidopsis, interact with the translational machinery.

2017-12-31 | GSE99826 | GEO

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Cotranslational signal independent SRP preloading during membrane targeting

Project description:Cotranslational targeting into the endoplasmic reticulum (ER) by the Signal Recognition Particle (SRP) is a key event determining polypeptide fate in eukaryotic cells. Here, we globally define the principles and mechanisms of SRP binding and ER targeting in vivo. Cotranslational targeting through SRP is the dominant route into the ER for all secretory proteins, regardless of targeting signal characteristics. Cytosolic SRP functions in a pioneer translation round that builds a membrane-resident mRNAs pool, explaining how low SRP levels suffice for the secretory load. SRP does not induce an elongation arrest; consequently, kinetic competition between targeting and translation elongation dictates which substrates are translocated post-translationally. Unexpectedly, SRP binds most secretory ribosomal complexes before targeting signals are synthesized. We show non-coding mRNA elements can promote signal-independent SRP pre-recruitment. Our study defines the complex kinetic interplay between elongation and determinants in the polypeptide and mRNA modulating SRP-substrate selection and membrane targeting in vivo. Ribosome profiling (RiboSeq) and RNA-seq of subcellular fractions of ribosomes. Soluble and membrane bound ribosomes are separated by centrifugation, and SRP-bound ribosomes are immunoprecipitated from the soluble fraction. Polysomes and monosomes are separated by sucrose gradient ultracentrifugation.

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