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Identification of embryonic RNA granules that act as sites of mRNA translation after changing their physical properties.

ABSTRACT: Fertilized eggs begin to translate mRNAs at appropriate times and placements to control development, but how the translation is regulated remains unclear. Here, we found that pou5f3 mRNA encoding a transcriptional factor essential for development formed granules in a dormant state in zebrafish oocytes. Although the number of pou5f3 granules remained constant, Pou5f3 protein accumulated after fertilization. Intriguingly, signals of newly synthesized peptides and a ribosomal protein became colocalized with pou5f3 granules after fertilization and, moreover, nascent Pou5f3 was shown to be synthesized in the granules. This functional change was accompanied by changes in the state and internal structure of granules. Dissolution of the granules reduced the rate of protein synthesis. Similarly, nanog and sox19b mRNAs in zebrafish and Pou5f1/Oct4 mRNA in mouse assembled into granules. Our results reveal that subcellular compartments, termed embryonic RNA granules, function as activation sites of translation after changing physical properties for directing vertebrate development.

SUBMITTER: Sato K

PROVIDER: S-EPMC9127168 | biostudies-literature | 2022 Jun

REPOSITORIES: biostudies-literature

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Identification of embryonic RNA granules that act as sites of mRNA translation after changing their physical properties.

Sato Keisuke K Sakai Moeko M Ishii Anna A Maehata Kaori K Takada Yuki Y Yasuda Kyota K Kotani Tomoya T

iScience 20220505 6

Fertilized eggs begin to translate mRNAs at appropriate times and placements to control development, but how the translation is regulated remains unclear. Here, we found that <i>pou5f3</i> mRNA encoding a transcriptional factor essential for development formed granules in a dormant state in zebrafish oocytes. Although the number of <i>pou5f3</i> granules remained constant, Pou5f3 protein accumulated after fertilization. Intriguingly, signals of newly synthesized peptides and a ribosomal protein ...[more]

PMID: 35620421

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Project description:Local translation in neurons is partly mediated by the reactivation of stalled polysomes. Stalled polysomes may be enriched within the granule fraction, defined as the pellet of sucrose gradients used to separate polysomes from monosomes. The mechanism of how elongating ribosomes are reversibly stalled and unstalled on mRNAs is still unclear. In the present study, we characterize the ribosomes in the granule fraction using immunoblotting, cryogenic electron microscopy (cryo-EM), and ribosome profiling. We find that this fraction, isolated from 5-d-old rat brains of both sexes, is enriched in proteins implicated in stalled polysome function, such as the fragile X mental retardation protein (FMRP) and Up-frameshift mutation 1 homologue. Cryo-EM analysis of ribosomes in this fraction indicates they are stalled, mainly in the hybrid state. Ribosome profiling of this fraction reveals (1) an enrichment for footprint reads of mRNAs that interact with FMRPs and are associated with stalled polysomes, (2) an abundance of footprint reads derived from mRNAs of cytoskeletal proteins implicated in neuronal development, and (3) increased ribosome occupancy on mRNAs encoding RNA binding proteins. Compared with those usually found in ribosome profiling studies, the footprint reads were longer and were mapped to reproducible peaks in the mRNAs. These peaks were enriched in motifs previously associated with mRNAs cross-linked to FMRP in vivo, independently linking the ribosomes in the granule fraction to the ribosomes associated with FMRP in the cell. The data supports a model in which specific sequences in mRNAs act to stall ribosomes during translation elongation in neurons.SIGNIFICANCE STATEMENT Neurons send mRNAs to synapses in RNA granules, where they are not translated until an appropriate stimulus is given. Here, we characterize a granule fraction obtained from sucrose gradients and show that polysomes in this fraction are stalled on consensus sequences in a specific state of translational arrest with extended ribosome-protected fragments. This finding greatly increases our understanding of how neurons use specialized mechanisms to regulate translation and suggests that many studies on neuronal translation may need to be re-evaluated to include the large fraction of neuronal polysomes found in the pellet of sucrose gradients used to isolate polysomes.

Dataset Information

Identification of embryonic RNA granules that act as sites of mRNA translation after changing their physical properties.

Publications

Identification of embryonic RNA granules that act as sites of mRNA translation after changing their physical properties.

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