RESUMO
Most eukaryotic mRNAs harbor a characteristic 5' m7GpppN cap that promotes pre-mRNA splicing, mRNA nucleocytoplasmic transport and translation while also protecting mRNAs from exonucleolytic attacks. mRNA caps are eliminated by Dcp2 during mRNA decay, allowing 5'-3' exonucleases to degrade mRNA bodies. However, the Dcp2 decapping enzyme is poorly active on its own and requires binding to stable or transient protein partners to sever the cap of target mRNAs. Here, we analyse the role of one of these partners, the yeast Pby1 factor, which is known to co-localize into P-bodies together with decapping factors. We report that Pby1 uses its C-terminal domain to directly bind to the decapping enzyme. We solved the structure of this Pby1 domain alone and bound to the Dcp1-Dcp2-Edc3 decapping complex. Structure-based mutant analyses reveal that Pby1 binding to the decapping enzyme is required for its recruitment into P-bodies. Moreover, Pby1 binding to the decapping enzyme stimulates growth in conditions in which decapping activation is compromised. Our results point towards a direct connection of Pby1 with decapping and P-body formation, both stemming from its interaction with the Dcp1-Dcp2 holoenzyme.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Endorribonucleases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Trifosfato de Adenosina/metabolismo , Domínio Catalítico , Proteínas de Ligação a DNA/química , Endopeptidases/química , Endopeptidases/metabolismo , Endorribonucleases/química , Holoenzimas/química , Holoenzimas/metabolismo , Ligases/metabolismo , Modelos Moleculares , Organelas/enzimologia , Organelas/metabolismo , Ligação Proteica , Domínios Proteicos , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/química , Fatores de Transcrição/químicaRESUMO
The Pat1 protein is a central player of eukaryotic mRNA decay that has also been implicated in translational control. It is commonly considered a central platform responsible for the recruitment of several RNA decay factors. We demonstrate here that a yeast-specific C-terminal region from Pat1 interacts with several short motifs, named helical leucine-rich motifs (HLMs), spread in the long C-terminal region of yeast Dcp2 decapping enzyme. Structures of Pat1-HLM complexes reveal the basis for HLM recognition by Pat1. We also identify a HLM present in yeast Xrn1, the main 5'-3' exonuclease involved in mRNA decay. We show further that the ability of yeast Pat1 to bind HLMs is required for efficient growth and normal mRNA decay. Overall, our analyses indicate that yeast Pat1 uses a single binding surface to successively recruit several mRNA decay factors and show that interaction between those factors is highly polymorphic between species.
Assuntos
Endorribonucleases/metabolismo , Exorribonucleases/metabolismo , Proteínas Fúngicas/metabolismo , RNA Mensageiro/metabolismo , Leveduras/metabolismo , Ligação Proteica/fisiologia , Domínios Proteicos/fisiologia , Estabilidade de RNA/fisiologia , Proteínas de Ligação a RNA/metabolismoRESUMO
Elimination of the 5' cap of eukaryotic mRNAs, known as decapping, is considered to be a crucial, irreversible and highly regulated step required for the rapid degradation of mRNA by Xrn1, the major cytoplasmic 5'-3' exonuclease. Decapping is accomplished by the recruitment of a protein complex formed by the Dcp2 catalytic subunit and its Dcp1 cofactor. However, this complex has a low intrinsic enzymatic activity and requires several accessory proteins such as the Lsm1-7 complex, Pat1, Edc1-Edc2 and/or Edc3 to be fully active. Here we present the crystal structure of the active form of the yeast Kluyveromyces lactis Dcp1-Dcp2 enzyme bound to its product (m7GDP) and its potent activator Edc3. This structure of the Dcp1-Dcp2 complex bound to a cap analog further explains previously published data on substrate binding and provides hints as to the mechanism of Edc3-mediated Dcp2 activation.