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MRNA localization to P-bodies in yeast is bi-phasic with many mRNAs captured in a late Bfr1p-dependent wave.

ABSTRACT: The relocalization of translationally repressed mRNAs to mRNA processing bodies Pbodies is a key consequence of cellular stress across many systems. Pbodies harbor mRNA degradation components and are implicated in mRNA decay, but the relative timing and control of mRNA relocalization to Pbodies is poorly understood. We used the MS2GFP system to follow the movement of specific endogenous mRNAs in live Saccharomyces cerevisiae cells after nutritional stress. It appears that the relocalization of mRNA to Pbodies after stress is biphasic some mRNAs are present early, whereas others are recruited much later concomitant with recruitment of translation initiation factors, such as eIF4E. We also find that Bfr1p is a latephaselocalizing Pbody protein that is important for the delayed entry of certain mRNAS to Pbodies. Therefore, for the mRNAs tested, relocalization to Pbodies varies both in terms of the kinetics and factor requirements. This work highlights a potential new regulatory juncture in gene expression that would facilitate the overall rationalization of protein content required for adaptation to stress.

SUBMITTER: Simpson CE

PROVIDER: S-EPMC3953815 | biostudies-other | 2014 Mar

REPOSITORIES: biostudies-other

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mRNA localization to P-bodies in yeast is bi-phasic with many mRNAs captured in a late Bfr1p-dependent wave.

Simpson Clare E CE Lui Jennifer J Kershaw Christopher J CJ Sims Paul F G PF Ashe Mark P MP

Journal of cell science 20140114 Pt 6

The relocalization of translationally repressed mRNAs to mRNA processing bodies Pbodies is a key consequence of cellular stress across many systems. Pbodies harbor mRNA degradation components and are implicated in mRNA decay, but the relative timing and control of mRNA relocalization to Pbodies is poorly understood. We used the MS2GFP system to follow the movement of specific endogenous mRNAs in live Saccharomyces cerevisiae cells after nutritional stress. It appears that the relocalization of m ...[more]

PMID: 24424022

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Project description:Orthopneumoviruses characteristically form membrane-less cytoplasmic inclusion bodies (IBs) wherein RNA replication and transcription occur. Here, we report a strategy whereby the orthopneumoviruses sequester various components of the translational preinitiation complex machinery into viral inclusion bodies to facilitate translation of their own mRNAs-PIC-pocketing. Electron microscopy of respiratory syncytial virus (RSV)-infected cells revealed bi-phasic organization of IBs, specifically, spherical "droplets" nested within the larger inclusion. Using correlative light and electron microscopy, combined with fluorescence in situ hybridization, we showed that the observed bi-phasic morphology represents functional compartmentalization of the inclusion body and that these domains are synonymous with the previously reported inclusion body-associated granules (IBAGs). Detailed analysis demonstrated that IBAGs concentrate nascent viral mRNA, the viral M2-1 protein as well as components of eukaryotic translation initiation factors (eIF), eIF4F and eIF3, and 40S complexes involved in translation initiation. Interestingly, although ribopuromycylation-based imaging indicates that the majority of viral mRNA translation occurs in the cytoplasm, there was some evidence for intra-IBAG translation, consistent with the likely presence of ribosomes in a subset of IBAGs imaged by electron microscopy. Mass spectrometry analysis of sub-cellular fractions from RSV-infected cells identified significant modification of the cellular translation machinery; however, interestingly, ribopuromycylation assays showed no changes to global levels of translation. The mechanistic basis for this pathway was subsequently determined to involve the viral M2-1 protein interacting with eIF4G, likely to facilitate its transport between the cytoplasm and the separate phases of the viral inclusion body. In summary, our data show that these viral organelles function to spatially regulate early steps in viral translation within a highly selective bi-phasic biomolecular condensate.ImportanceRespiratory syncytial viruses (RSVs) of cows and humans are a significant cause of morbidity and mortality in their respective populations. These RNA viruses replicate in the infected cells by compartmentalizing the cell's cytoplasm into distinct viral microdomains called inclusion bodies (IBs). In this paper, we show that these IBs are further compartmentalized into smaller structures that have significantly different density, as observed by electron microscopy. Within smaller intra-IB structures, we observed ribosomal components and evidence for active translation. These findings highlight that RSV may additionally compartmentalize translation to favor its own replication in the cell. These data contribute to our understanding of how RNA viruses hijack the cell to favor replication of their own genomes and may provide new targets for antiviral therapeutics in vivo.

Dataset Information

MRNA localization to P-bodies in yeast is bi-phasic with many mRNAs captured in a late Bfr1p-dependent wave.

Publications

mRNA localization to P-bodies in yeast is bi-phasic with many mRNAs captured in a late Bfr1p-dependent wave.

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