The 5’→3’ exoribonucleases (XRNs) comprise a large family of conserved enzymes in eukaryotes with crucial functions in RNA metabolism and RNA interference1–5. XRN2, or Rat1 in yeast6, functions primarily in the nucleus and also plays an important role in transcription termination by RNA polymerase II (Pol II)7–14. Rat1 exoribonuclease activity is stimulated by the protein Rai115, 16. Here we report the crystal structure at 2.2 Å resolution of S. pombe Rat1 in complex with Rai1, as well as the structures of Rai1 and its murine homolog DOM3Z alone at 2.0 Å 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 more effectively degrade RNAs with stable secondary structure. There are large differences in the active site landscape of Rat1 compared to related and PIN (PilT N-terminus) domain-containing nucleases17–20. 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 mRNA degradation in bacteria21, but ours is the first demonstration of this activity in eukaryotes and suggests that Rai1/DOM3Z may have additional important functions in RNA metabolism.

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.

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.The cytoplasmic polyadenylation element binding (CPEB) protein, a sequence-specific RNA binding protein, is found in the cell body and at synapses of hippocampal and other neurons; in response to synaptic experience, CPEB promotes polyadenylation and translation (37,49,50). CPEB knockout mice (44) have deficiencies in synaptic plasticity, particularly, theta burst-induced long-term potentiation (LTP) (1, 51), as well as in particular forms of hippocampal-dependent memories (3). Some of these CPEB-related functions may be related at least in part to its ability to direct CPE-containing RNA transport in dendrites (16), in addition to its regulation of translation (51).In neurons, most of the CPEB-related proteins CPEB3 and CPEB4 are found in the cell soma. However, a relatively small amount of these proteins is localized to synaptic regions and cofractionates with postsynaptic density (PSD) proteins, suggesting possible roles in RNA translation and/or localization (17). While investigating the possible translocation of CPEB3 and -4 to dendritic spines in response to N-methyl-D-aspartate receptor (NMDAR) activation, as is the case with the fragile X mental retardation protein (FMRP) (11), we noticed a surprising relocalization of CPEB4 from the cell soma to the nucleus. While the treatment of neurons with ionotropic glutamate receptor-activating agents, such as glutamate and ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), as well as NMDA, induced nuclear localization of CPEB4, 3,5-dihydroxyphenylglycine (DHPG), an agonist of the metabotropic glutamate receptors, did not. CPEB4 remained cytoplasmic when calcium/calmodulin-dependent protein kinase II (CaMKII) activity was inhibited, suggesting a link between calcium levels and nuclear i...

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