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A receptor for activated C kinase is part of messenger ribonucleoprotein complexes associated with polyA-mRNAs in neurons.

ABSTRACT: Long-lasting changes in synaptic functions after an appropriate stimulus require altered protein expression at the synapse. To restrict changes in protein composition to activated synapses, proteins may be synthesized locally as a result of transmitter receptor-triggered signaling pathways. Second messenger-controlled mechanisms that affect mRNA translation are essentially unknown. Here we report that a receptor for activated C kinase, RACK1, is a component of messenger ribonucleoprotein (mRNP) complexes. RACK1 is predominantly associated with polysome-bound, polyA-mRNAs that are being actively translated. We find it to be present in a complex with beta-tubulin and at least two mRNA-binding proteins, polyA-binding protein 1 and a 130 kDa polyA-mRNA binding protein (KIAA0217). Activation of PKCbeta2 in vitro by phosphatidylserine/diacylglycerol or in hippocampal slices by metabotropic glutamate receptor stimulation increased the amount of RACK1/PKCbeta2 associated with polysome-bound polyA-mRNAs. In vitro, PKCbeta2 can phosphorylate a subset of polyA-mRNA-associated proteins that are also phosphorylated under in vivo conditions. On the basis of these findings plus the somatodendritic localization of RACK1, we hypothesize that metabotropic glutamate receptor-triggered binding of activated PKCbeta2 to mRNP complexes bound to polyA-mRNAs is involved in activity-triggered control of protein synthesis.

SUBMITTER: Angenstein F

PROVIDER: S-EPMC6757688 | biostudies-literature | 2002 Oct

REPOSITORIES: biostudies-literature

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A receptor for activated C kinase is part of messenger ribonucleoprotein complexes associated with polyA-mRNAs in neurons.

Angenstein Frank F Evans Anne M AM Settlage Robert E RE Moran Stewart T ST Ling Shuo-Chien SC Klintsova Anna Y AY Shabanowitz Jeffrey J Hunt Donald F DF Greenough William T WT

The Journal of neuroscience : the official journal of the Society for Neuroscience 20021001 20

Long-lasting changes in synaptic functions after an appropriate stimulus require altered protein expression at the synapse. To restrict changes in protein composition to activated synapses, proteins may be synthesized locally as a result of transmitter receptor-triggered signaling pathways. Second messenger-controlled mechanisms that affect mRNA translation are essentially unknown. Here we report that a receptor for activated C kinase, RACK1, is a component of messenger ribonucleoprotein (mRNP) ...[more]

PMID: 12388589

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Project description:The 3'-UTR (untranslated region) of bcl-2 mRNA contains an ARE (AU-rich element) that potentially regulates the stability of bcl-2 mRNA in a cell specific fashion. Previous studies have demonstrated that multiple proteins interact with bcl-2 mRNA in HL-60 (human leukaemia-60) cells, potentially contributing to the overexpression of Bcl-2 protein. Treatment of HL-60 cells with taxol or okadaic acid has been shown to induce destabilization of bcl-2 mRNA, which was associated with decreased binding of trans-acting factors to bcl-2 mRNA. Nucleolin has been identified as one of the bcl-2 mRNA-binding proteins [Sengupta, Bandyopadhyay, Fernandes and Spicer (2004) J. Biol. Chem. 279, 10855-10863]. In an effort to identify additional bcl-2 mRNA-binding proteins, two polypeptides of approx. 45 kDa and 60 kDa were isolated from HL-60 cells by ARE(bcl-2) (transcripts that contain bcl-2 AREs) RNA affinity chromatography. These proteins were identified as the human proliferation associated protein, Ebp1, and human DRBP76 (double stranded RNA-binding protein 76) respectively, by MALDI (matrix-assisted laser-desorption ionization)-MS. RNA electrophoretic mobility shift assays indicated that recombinant Ebp1 binds to ARE(bcl-2) RNA but not to the group 1 ARE present in GM-CSF (granulocyte macrophage-colony stimulating factor) mRNA in vitro. Antibody supershift assays demonstrated that Ebp1 is present in protein-ARE(bcl-2) RNA complexes formed with cytosolic HL-60 extracts. The interaction of Ebp1 with bcl-2 mRNA in HL-60 cells was also demonstrated by RNA co-immunoprecipitation assays. This interaction was not detected in extracts of taxol-treated HL-60 cells. Immunoprecipitation assays further revealed that Ebp1 co-precipitates with nucleolin from HL-60 cytoplasmic extracts. The observation that co-precipitation was decreased when extracts were treated with RNase suggests that Ebp1 and nucleolin are present in the same bcl-2 mRNP (messenger ribonucleoprotein particle) complexes. RNA-decay assays further demonstrated that Ebp1 decreased the rate of decay of beta-globin-ARE(bcl-2) transcripts in HL-60 cell extracts. Collectively, these results indicate a novel function for Ebp1 in contributing to the regulation of bcl-2 expression in HL-60 cells.

Project description:BackgroundPlatelets are small anucleate cells circulating in the blood vessels where they play a key role in hemostasis and thrombosis. Here, we compared platelet RNA-Seq results obtained from polyA+ mRNA and rRNA-depleted total RNA.Materials and methodsWe used purified, CD45 depleted, human blood platelets collected by apheresis from three male and one female healthy blood donors. The Illumina HiSeq 2000 platform was employed to sequence cDNA converted either from oligo(dT) isolated polyA+ RNA or from rRNA-depleted total RNA. The reads were aligned to the GRCh37 reference assembly with the TopHat/Cufflinks alignment package using Ensembl annotations. A de novo assembly of the platelet transcriptome using the Trinity software package and RSEM was also performed. The bioinformatic tools HTSeq and DESeq from Bioconductor were employed for further statistical analyses of read counts.ResultsConsistent with previous findings our data suggests that mitochondrially expressed genes comprise a substantial fraction of the platelet transcriptome. We also identified high transcript levels for protein coding genes related to the cytoskeleton function, chemokine signaling, cell adhesion, aggregation, as well as receptor interaction between cells. Certain transcripts were particularly abundant in platelets compared with other cell and tissue types represented by RNA-Seq data from the Illumina Human Body Map 2.0 project. Irrespective of the different library preparation and sequencing protocols, there was good agreement between samples from the 4 individuals. Eighteen differentially expressed genes were identified in the two sexes at 10% false discovery rate using DESeq.ConclusionThe present data suggests that platelets may have a unique transcriptome profile characterized by a relative over-expression of mitochondrially encoded genes and also of genomic transcripts related to the cytoskeleton function, chemokine signaling and surface components compared with other cell and tissue types. The in vivo functional significance of the non-mitochondrial transcripts remains to be shown.

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A receptor for activated C kinase is part of messenger ribonucleoprotein complexes associated with polyA-mRNAs in neurons.

Publications

A receptor for activated C kinase is part of messenger ribonucleoprotein complexes associated with polyA-mRNAs in neurons.

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