RESUMEN
The RNA binding protein CPEB (cytoplasmic polyadenylation element binding) regulates cytoplasmic polyadenylation and translation in germ cells and the brain. In neurons, CPEB is detected at postsynaptic sites, as well as in the cell body. The related CPEB3 protein also regulates translation in neurons, albeit probably not through polyadenylation; it, as well as CPEB4, is present in dendrites and the cell body. Here, we show that treatment of neurons with ionotropic glutamate receptor agonists causes CPEB4 to accumulate in the nucleus. All CPEB proteins are nucleus-cytoplasm shuttling proteins that are retained in the nucleus in response to calcium-mediated signaling and alpha-calcium/calmodulin-dependent kinase protein II (CaMKII) activity. CPEB2, -3, and -4 have conserved nuclear export signals that are not present in CPEB. CPEB4 is necessary for cell survival and becomes nuclear in response to focal ischemia in vivo and when cultured neurons are deprived of oxygen and glucose. Further analysis indicates that nuclear accumulation of CPEB4 is controlled by the depletion of calcium from the ER, specifically, through the inositol-1,4,5-triphosphate (IP3) receptor, indicating a communication between these organelles in redistributing proteins between subcellular compartments.
Asunto(s)
Isquemia Encefálica/metabolismo , Calcio/metabolismo , Núcleo Celular/metabolismo , Retículo Endoplásmico/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas de Unión al ARN/metabolismo , Secuencia de Aminoácidos , Animales , Células Cultivadas , Humanos , Datos de Secuencia Molecular , N-Metilaspartato/farmacología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Isoformas de Proteínas/genética , Proteínas de Unión al ARN/genética , Ratas , Receptores Ionotrópicos de Glutamato/agonistas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Bloqueadores de los Canales de Sodio/farmacología , Tetrodotoxina/farmacologíaRESUMEN
The 5'-->3' exoribonucleases (XRNs) comprise a large family of conserved enzymes in eukaryotes with crucial functions in RNA metabolism and RNA interference. XRN2, or Rat1 in yeast, functions primarily in the nucleus and also has an important role in transcription termination by RNA polymerase II (refs 7-14). Rat1 exoribonuclease activity is stimulated by the protein Rai1 (refs 15, 16). Here we report the crystal structure at 2.2 A resolution of Schizosaccharomyces pombe Rat1 in complex with Rai1, as well as the structures of Rai1 and its murine homologue Dom3Z alone at 2.0 A resolution. The structures reveal the molecular mechanism for the activation of Rat1 by Rai1 and for the exclusive exoribonuclease activity of Rat1. Biochemical studies confirm these observations, and show that Rai1 allows Rat1 to degrade RNAs with stable secondary structure more effectively. There are large differences in the active site landscape of Rat1 compared to related and PIN (PilT N terminus) domain-containing nucleases. Unexpectedly, we identified a large pocket in Rai1 and Dom3Z that contains highly conserved residues, including three acidic side chains that coordinate a divalent cation. Mutagenesis and biochemical studies demonstrate that Rai1 possesses pyrophosphohydrolase activity towards 5' triphosphorylated RNA. Such an activity is important for messenger RNA degradation in bacteria, but this is, to our knowledge, the first demonstration of this activity in eukaryotes and suggests that Rai1/Dom3Z may have additional important functions in RNA metabolism.
Asunto(s)
Exorribonucleasas/química , Exorribonucleasas/metabolismo , Modelos Moleculares , Proteínas Nucleares , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces , Animales , Exorribonucleasas/genética , Ratones , Mutación , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Schizosaccharomyces/química , Schizosaccharomyces/enzimología , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genéticaRESUMEN
Tbx6 is a member of the T-box family of transcription factor genes. Two mutant alleles of this gene establish that Tbx6 is involved in both the specification and patterning of the somites along the entire length of the embryo. The null allele, Tbx6(tm1Pa), causes abnormal patterning of the cervical somites and improper specification of more posterior paraxial mesoderm, such that it forms ectopic neural tubes. In this study, we use this allele to further investigate the mechanism of action of the Tbx6 gene and investigate possible genetic interactions. We have tested the developmental and differentiation potential of Tbx6(tm1Pa)/Tbx6(tm1Pa) cells in ectopic sites, in vitro, and in chimeras in vivo. We have also documented cell proliferation and cell death in mutant tail buds in an attempt to explain the mechanism of tail bud enlargement in the Tbx6 mutant embryos. Our results indicate specific developmental restrictions on the differentiation of posterior cells lacking Tbx6, once they have traversed the primitive streak, but no restrictions in differentiation of anterior somites, or of Tbx6 null embryonic stem (ES) cells. We further demonstrate that Tbx6 null ES cells fail to populate posterior somites in chimeric embryos. To discover whether different T-box proteins interact on the same down stream targets in areas of expression overlap, we have explored potential interactions between Tbx6 and T (Brachyury) in genetic crosses. Our results reveal that the T(Wis) mutation is epistatic to the Tbx6(tm1Pa) mutation and that there is no apparent genetic interaction. However, homozygosity for Tbx6(tm1Pa) and heterozygosity for T(Wis) mutation shows a combinatorial interaction at the phenotypic level.