Dcp2 phosphorylation by Ste20 modulates stress granule assembly and mRNA decay in Saccharomyces cerevisiae

Department of Molecular and Cellular Biology, University of Arizona, Tucson, AZ 85721, USA.
The Journal of Cell Biology (Impact Factor: 9.83). 05/2010; 189(5):813-27. DOI: 10.1083/jcb.200912019
Source: PubMed

ABSTRACT Translation and messenger RNA (mRNA) degradation are important sites of gene regulation, particularly during stress where translation and mRNA degradation are reprogrammed to stabilize bulk mRNAs and to preferentially translate mRNAs required for the stress response. During stress, untranslating mRNAs accumulate both in processing bodies (P-bodies), which contain some translation repressors and the mRNA degradation machinery, and in stress granules, which contain mRNAs stalled in translation initiation. How signal transduction pathways impinge on proteins modulating P-body and stress granule formation and function is unknown. We show that during stress in Saccharomyces cerevisiae, Dcp2 is phosphorylated on serine 137 by the Ste20 kinase. Phosphorylation of Dcp2 affects the decay of some mRNAs and is required for Dcp2 accumulation in P-bodies and specific protein interactions of Dcp2 and for efficient formation of stress granules. These results demonstrate that Ste20 has an unexpected role in the modulation of mRNA decay and translation and that phosphorylation of Dcp2 is an important control point for mRNA decapping.

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    • "Recombinant maltose-binding protein (MBP)-HuR was incubated with a buffer containing 20 mM Tris-HCl at pH 7.5, 100 mM KCl, 5 mM MgCl2 and 0.5% NP-40. Biotinylated SIRT1 and GAPDH 3′-untranslated regions were synthesized by PCR amplification of cDNA using forward primers that contained the T7 RNA polymerase promoter sequence (Supplementary Table S1) in the presence of biotinylated CTP and T7 RNA polymerase, as described (22,31). Proteins present in the pulldown material were studied by WB analysis. "
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    ABSTRACT: In response to stress conditions, many mammalian mRNAs accumulate in stress granules (SGs) together with numerous RNA-binding proteins that control mRNA turnover and translation. However, the signaling cascades that modulate the presence of ribonucleoprotein (RNP) complexes in SGs are poorly understood. Here, we investigated the localization of human antigen R (HuR), an mRNA-stabilizing RNA-binding protein, in SGs following exposure to the stress agent arsenite. Unexpectedly, the mobilization of HuR to SGs was prevented through the activation of Janus kinase 3 (JAK3) by the vitamin K3 analog menadione. JAK3 phosphorylated HuR at tyrosine 200, in turn inhibiting HuR localization in SGs, reducing HuR interaction with targets SIRT1 and VHL mRNAs, and accelerating target mRNA decay. Our findings indicate that HuR is tyrosine-phosphorylated by JAK3, and link this modification to HuR subcytoplasmic localization and to the fate of HuR target mRNAs.
    Nucleic Acids Research 10/2013; 42(2). DOI:10.1093/nar/gkt903 · 9.11 Impact Factor
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    • "However, a fraction of these deadenylated targets may be degraded through decapping when DCP2 and DCP1 are expressed. Additionally, DCP1 and DCP2 are phosphorylated under cellular stress conditions (47,48), and DCP1 is hyperphosphorylated during mitosis (49). Under these conditions, a subset of mRNAs is stabilized, suggesting that DCP1 and DCP2 phosphorylation inhibits decapping. "
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    ABSTRACT: MicroRNA (miRNA)-induced silencing complexes (miRISCs) repress translation and promote degradation of miRNA targets. Target degradation occurs through the 5'-to-3' messenger RNA (mRNA) decay pathway, wherein, after shortening of the mRNA poly(A) tail, the removal of the 5' cap structure by decapping triggers irreversible decay of the mRNA body. Here, we demonstrate that miRISC enhances the association of the decapping activators DCP1, Me31B and HPat with deadenylated miRNA targets that accumulate when decapping is blocked. DCP1 and Me31B recruitment by miRISC occurs before the completion of deadenylation. Remarkably, miRISC recruits DCP1, Me31B and HPat to engineered miRNA targets transcribed by RNA polymerase III, which lack a cap structure, a protein-coding region and a poly(A) tail. Furthermore, miRISC can trigger decapping and the subsequent degradation of mRNA targets independently of ongoing deadenylation. Thus, miRISC increases the local concentration of the decapping machinery on miRNA targets to facilitate decapping and irreversibly shut down their translation.
    Nucleic Acids Research 07/2013; 41(18). DOI:10.1093/nar/gkt619 · 9.11 Impact Factor
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    • "Thus, selective activation of decapping may potentially be a viable therapeutic approach to degrade RVFV-accessible mRNAs. Indeed, Dcp2 is potentially regulated: In yeast, phosphorylation of Dcp2 by Ste20 is necessary for Dcp2 recruitment to P bodies (Yoon et al. 2010), and vertebrate Dcp2 has a number of conserved uncharacterized phosphorylation sites (Hornbeck et al. 2011). Further studies will reveal whether decapping of specific cargo can be selectively regulated in insects and mammals. "
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    ABSTRACT: Bunyaviruses are an emerging group of medically important viruses, many of which are transmitted from insects to mammals. To identify host factors that impact infection, we performed a genome-wide RNAi screen in Drosophila and identified 131 genes that impacted infection of the mosquito-transmitted bunyavirus Rift Valley fever virus (RVFV). Dcp2, the catalytic component of the mRNA decapping machinery, and two decapping activators, DDX6 and LSM7, were antiviral against disparate bunyaviruses in both insect cells and adult flies. Bunyaviruses 5' cap their mRNAs by "cap-snatching" the 5' ends of poorly defined host mRNAs. We found that RVFV cap-snatches the 5' ends of Dcp2 targeted mRNAs, including cell cycle-related genes. Loss of Dcp2 allows increased viral transcription without impacting viral mRNA stability, while ectopic expression of Dcp2 impedes viral transcription. Furthermore, arresting cells in late S/early G2 led to increased Dcp2 mRNA targets and increased RVFV replication. Therefore, RVFV competes for the Dcp2-accessible mRNA pool, which is dynamically regulated and can present a bottleneck for viral replication.
    Genes & development 07/2013; 27(13):1511-25. DOI:10.1101/gad.215384.113 · 10.80 Impact Factor
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