RESUMO
In multicellular organisms, tight regulation of gene expression ensures appropriate tissue and organismal growth throughout development. Reversible phosphorylation of the RNA Polymerase II (RNAPII) C-terminal domain (CTD) is critical for the regulation of gene expression states, but how phosphorylation is actively modified in a developmental context remains poorly understood. Protein phosphatase 1 (PP1) is one of several enzymes that has been reported to dephosphorylate the RNAPII CTD. However, PP1's contribution to transcriptional regulation during animal development and the mechanisms by which its activity is targeted to RNAPII have not been fully elucidated. Here we show that the Drosophila orthologue of the PP1 Nuclear Targeting Subunit (dPNUTS) is essential for organismal development and is cell autonomously required for growth of developing tissues. The function of dPNUTS in tissue development depends on its binding to PP1, which we show is targeted by dPNUTS to RNAPII at many active sites of transcription on chromosomes. Loss of dPNUTS function or specific disruption of its ability to bind PP1 results in hyperphosphorylation of the RNAPII CTD in whole animal extracts and on chromosomes. Consistent with dPNUTS being a global transcriptional regulator, we find that loss of dPNUTS function affects the expression of the majority of genes in developing 1(st) instar larvae, including those that promote proliferative growth. Together, these findings shed light on the in vivo role of the PNUTS-PP1 holoenzyme and its contribution to the control of gene expression during early Drosophila development.
Assuntos
Drosophila melanogaster/genética , Proteína Fosfatase 1/biossíntese , RNA Polimerase II/genética , Transcrição Gênica , Animais , Domínio Catalítico/genética , Proteínas de Ligação a DNA/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/crescimento & desenvolvimento , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Dados de Sequência Molecular , Fosforilação/genética , Proteína Fosfatase 1/química , Proteína Fosfatase 1/genética , Estrutura Terciária de Proteína/genética , RNA Polimerase II/metabolismo , Proteínas de Ligação a RNA/genéticaRESUMO
BACKGROUND: Protein phosphatase 1 (PP1) is involved in diverse cellular processes, and is targeted to substrates via interaction with many different protein binding partners. PP1 catalytic subunits (PP1c) fall into PP1alpha and PP1beta subfamilies based on sequence analysis, however very few PP1c binding proteins have been demonstrated to discriminate between PP1alpha and PP1beta. RESULTS: URI (unconventional prefoldin RPB5 interactor) is a conserved molecular chaperone implicated in a variety of cellular processes, including the transcriptional response to nutrient signalling and maintenance of DNA integrity. We show that Drosophila Uri binds PP1alpha with much higher affinity than PP1beta, and that this ability to discriminate between PP1c forms is conserved to humans. Most Uri is cytoplasmic, however we found some protein associated with active RNAPII on chromatin. We generated a uri loss of function allele, and show that uri is essential for viability in Drosophila. uri mutants have transcriptional defects, reduced cell viability and differentiation in the germline, and accumulate DNA damage in their nuclei. CONCLUSION: Uri is the first PP1alpha specific binding protein to be described in Drosophila. Uri protein plays a role in transcriptional regulation. Activity of uri is required to maintain DNA integrity and cell survival in normal development.
Assuntos
Proteínas de Drosophila/fisiologia , Chaperonas Moleculares/fisiologia , Proteína Fosfatase 1/fisiologia , Animais , Células COS , Diferenciação Celular/fisiologia , Sobrevivência Celular/fisiologia , Chlorocebus aethiops , Citoplasma/metabolismo , Dano ao DNA , Drosophila melanogaster/crescimento & desenvolvimento , Feminino , Humanos , Masculino , Ovário/metabolismo , Testículo/metabolismoRESUMO
Protein phosphatase Y (PPY) is a Drosophila testis-specific enzyme of unknown function. In a yeast two-hybrid screen we identified CG15031/PPYR1 as a PPY interacting protein. The specificity of the protein-protein interaction was proven by directed two-hybrid tests. The complex formation between PPY and PPYR1 was confirmed under in vitro and in vivo conditions by plasmon resonance spectroscopy, co-immunoprecipitation, and pull down experiments. Recombinant PPYR1 expressed in Escherichia coli is a heatstable, protease sensitive, intrinsically unstructured RNA-binding protein that migrates anomalously in SDS-polyacrylamide gel electrophoresis. It can be phosphorylated by cAMP-dependent protein kinase in vitro. PPYR1 moderately inhibits PPY activity, the inhibitory potential of the protein is slightly increased by phosphorylation. We suggest that PPYR1 may function as a scaffolding protein that targets PPY to RNA and other protein partners in Drosophila melanogaster.
Assuntos
Proteínas de Drosophila/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Receptores de Neuropeptídeo Y/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Fosfoproteínas Fosfatases/genética , Fosforilação , Proteínas de Ligação a RNA/metabolismo , Receptores de Neuropeptídeo Y/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ressonância de Plasmônio de SuperfícieRESUMO
Reversible phosphorylation of myosin regulatory light chain (MRLC) is a key regulatory mechanism controlling myosin activity and thus regulating the actin/myosin cytoskeleton. We show that Drosophila PP1beta, a specific isoform of serine/threonine protein phosphatase 1 (PP1), regulates nonmuscle myosin and that this is the essential role of PP1beta. Loss of PP1beta leads to increased levels of phosphorylated nonmuscle MRLC (Sqh) and actin disorganisation; these phenotypes can be suppressed by reducing the amount of active myosin. Drosophila has two nonmuscle myosin targeting subunits, one of which (MYPT-75D) resembles MYPT3, binds specifically to PP1beta, and activates PP1beta's Sqh phosphatase activity. Expression of a mutant form of MYPT-75D that is unable to bind PP1 results in elevation of Sqh phosphorylation in vivo and leads to phenotypes that can also be suppressed by reducing the amount of active myosin. The similarity between fly and human PP1beta and MYPT genes suggests this role may be conserved.
