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Identification of protein-protein interactions involving the eIF4E binding proteins Caf20p and Eap1p on yeast via affinity purification and LC-MSMS.

ABSTRACT: There are multiple translational control pathways. These include pathways involving eIF4E binding proteins (4E-BPs), which inhibit translation by binding and sequestering the 5’ cap binding protein eIF4E away from its partner eIF4G. Saccharomyces cerevisiae has two 4E-BPs: Caf20p and Eap1p. Previous analyses had shown that each 4E-BP regulates different subsets of mRNAs. In order to assess whether binding different proteins caused their different regulatory role, we used tandem affinity purification followed by label-free mass spectrometry to compare the proteomes pulled-down with the TAP-tagged 4E-BPs, and the global proteome. These analyses point out that Caf20p and Eap1p share most interaction partners, including ribosomes, and that both bind several other RNA-binding proteins.

INSTRUMENT(S): Dionex instrument model, Progenesis LC-MS, Mascot, LTQ Orbitrap Elite

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: David Talavera

LAB HEAD: Graham Pavitt

PROVIDER: PXD001407 | Pride | 2016-06-29

REPOSITORIES: Pride

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Publications

The 4E-BP Caf20p Mediates Both eIF4E-Dependent and Independent Repression of Translation.

Castelli Lydia M LM Talavera David D Kershaw Christopher J CJ Mohammad-Qureshi Sarah S SS Costello Joseph L JL Rowe William W Sims Paul F G PF Grant Christopher M CM Hubbard Simon J SJ Ashe Mark P MP Pavitt Graham D GD

PLoS genetics 20150514 5

Translation initiation factor eIF4E mediates mRNA selection for protein synthesis via the mRNA 5'cap. A family of binding proteins, termed the 4E-BPs, interact with eIF4E to hinder ribosome recruitment. Mechanisms underlying mRNA specificity for 4E-BP control remain poorly understood. Saccharomyces cerevisiae 4E-BPs, Caf20p and Eap1p, each regulate an overlapping set of mRNAs. We undertook global approaches to identify protein and RNA partners of both 4E-BPs by immunoprecipitation of tagged prot ...[more]

PMID: 25973932

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Project description:Virus infection may shut off host protein synthesis in order to achieve the replicative advantage over host cells. It is well known that human pathogenic viruses, particularly the picornaviruses, can block host protein synthesis by cleavage or inhibition of eukaryotic initiation factors (eIFs). In this study we found a novel mechanism that microRNA (miRNA) is involved in viral pathogenesis. Infection of enteroviruses can disturb the expression of host miRNAs, in which miR-141 is up-regulated and inhibits host protein synthesis by post-transcriptional repression of the target gene eIF4E, a key element for cap-dependent translation of host proteins. Knockdown of miR-141 by a specific siRNA, antagomiR-141, could restore host eIF4E expression, delay the occurrence of cytopathic effect (CPE), and impair virus propagation. We demonstrated that EV71 infection could increase early growth response 1 (EGR1) expression which induced miR-141 causing the eIF4E suppression; while silencing of EGR1 attenuated virus production. Our results suggest that enterovirus infection causes the EGR1-mediated upregulation of host miR-141, further lead to the translational switch from cap-dependent to cap-independent protein synthesis in the host cells, an environmental beneficial for viral propagation. This novel mechanism may highlight a new approach for future development of antiviral therapy. Enteroviruses in the Picornaviridae family are important human pathogens which can cause fatal diseases, including cardiopulmonary failure, aseptic meningitis, paralysis, myocarditis, and encephalomyelitis. Virus infection may induce shutoff of host protein synthesis, particularly in picornavirus, whose protein translation is cap-independent. It is known that poliovirus 2A protease cleaves eIF4G, a scaffold component of mammalian cell translational complex, leading to the shut down of host protein synthesis. Nevertheless, the cleavage of eIF4G may not be sufficient for the complete shutoff of host protein synthesis. Previous studies showed that cleavage of polyA-binding protein (PABP) by viral protease 3C and dephosphorylation of the translational repressor, eIF4E binding protein 1 (4E-BP1), also contribute to this process. The cap-binding protein, eIF4E, is the most crucial factor in determining whether cap-dependent or -independent translation takes place. The mechanism by which viral infection modulates host cell protein synthesis through interfering eIF4E expression is not yet known. miRNAs are a newly discovered class of small non-protein-coding RNAs that may act via endogenous RNA interference. Our understanding of its role in the dynamic interplay between virus and host components is quite limited. Since both virus infection and miRNAs could hinder cellular protein synthesis, whether miRNAs are involved during virus infection in shutting off host protein synthesis is still unknown. To address this issue, we analyze the altered gene and microRNA expression after EV71 infection. ***This submission represents the mRNA expression component of the study only***

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