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A circadian clock translational control mechanism targets specific mRNAs to cytoplasmic messenger ribonucleoprotein granules.

ABSTRACT: Phosphorylation of Neurospora crassa eukaryotic initiation factor 2 α (eIF2α), a conserved translation initiation factor, is clock controlled. To determine the impact of rhythmic eIF2α phosphorylation on translation, we performed temporal ribosome profiling and RNA sequencing (RNA-seq) in wild-type (WT), clock mutant Δfrq, eIF2α kinase mutant Δcpc-3, and constitutively active cpc-3^c cells. About 14% of mRNAs are rhythmically translated in WT cells, and translation rhythms for ∼30% of these mRNAs, which we named circadian translation-initiation-controlled genes (cTICs), are dependent on the clock and CPC-3. Most cTICs are expressed from arrhythmic mRNAs and contain a P-body (PB) localization motif in their 5' leader sequence. Deletion of SNR-1, a component of cytoplasmic messenger ribonucleoprotein granules (cmRNPgs) that include PBs and stress granules (SGs), and the PB motif on one of the cTIC mRNAs, zip-1, significantly alters zip-1 rhythmic translation. These results reveal that the clock regulates rhythmic translation of specific mRNAs through rhythmic eIF2α activity and cmRNPg metabolism.

SUBMITTER: Castillo KD

PROVIDER: S-EPMC10241597 | biostudies-literature | 2022 Dec

REPOSITORIES: biostudies-literature

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A circadian clock translational control mechanism targets specific mRNAs to cytoplasmic messenger ribonucleoprotein granules.

Castillo Kathrina D KD Wu Cheng C Ding Zhaolan Z Lopez-Garcia Osiris K OK Rowlinson Emma E Sachs Matthew S MS Bell-Pedersen Deborah D

Cell reports 20221201 13

Phosphorylation of Neurospora crassa eukaryotic initiation factor 2 α (eIF2α), a conserved translation initiation factor, is clock controlled. To determine the impact of rhythmic eIF2α phosphorylation on translation, we performed temporal ribosome profiling and RNA sequencing (RNA-seq) in wild-type (WT), clock mutant Δfrq, eIF2α kinase mutant Δcpc-3, and constitutively active cpc-3<sup>c</sup> cells. About 14% of mRNAs are rhythmically translated in WT cells, and translation rhythms for ∼30% of ...[more]

PMID: 36577368

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Project description:Transcriptome analyses indicate that a core 10%-15% of the yeast genome is modulated by a variety of different stresses. However, not all the induced genes undergo translation, and null mutants of many induced genes do not show elevated sensitivity to the particular stress. Elucidation of the RNA lifecycle reveals accumulation of non-translating mRNAs in cytoplasmic granules, P-bodies, and stress granules for future regulation. P-bodies contain enzymes for mRNA degradation; under stress conditions mRNAs may be transferred to stress granules for storage and return to translation. Protein degradation by the ubiquitin-proteasome system is elevated by stress; and here we analyzed the steady state levels, decay, and subcellular localization of the mRNA of the gene encoding the F-box protein, UFO1, that is induced by stress. Using the MS2L mRNA reporter system UFO1 mRNA was observed in granules that colocalized with P-bodies and stress granules. These P-bodies stored diverse mRNAs. Granules of two mRNAs transported prior to translation, ASH1-MS2L and OXA1-MS2L, docked with P-bodies. HSP12 mRNA that gave rise to highly elevated protein levels was not observed in granules under these stress conditions. ecd3, pat1 double mutants that are defective in P-body formation were sensitive to mRNAs expressed ectopically from strong promoters. These highly expressed mRNAs showed elevated translation compared with wild-type cells, and the viability of the mutants was strongly reduced. ecd3, pat1 mutants also exhibited increased sensitivity to different stresses. Our interpretation is that sequestration of highly expressed mRNAs in P-bodies is essential for viability. Storage of mRNAs for future regulation may contribute to the discrepancy between the steady state levels of many stress-induced mRNAs and their proteins. Sorting of mRNAs for future translation or decay by individual cells could generate potentially different phenotypes in a genetically identical population and enhance its ability to withstand stress.

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.

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A circadian clock translational control mechanism targets specific mRNAs to cytoplasmic messenger ribonucleoprotein granules.

Publications

A circadian clock translational control mechanism targets specific mRNAs to cytoplasmic messenger ribonucleoprotein granules.

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