Masumi Shimada

Novel family of CCCH-type zinc-finger proteins, MOE-1, -2 and -3, participates in C. elegans oocyte maturation.

Background: Oocyte maturation is an important prerequisite for the production of progeny. Although several germ‐line mutations have been reported, the precise mechanism by which the last step of oocyte maturation is controlled remains unclear. In Caenorhabditis elegans, CCCH‐type zinc‐finger proteins have been shown to be involved in germ cell formation, although their involvement in oocyte maturation has not been fully investigated.

Unique proteasome subunit Xrpn10c is a specific receptor for the antiapoptotic ubiquitin‐like protein Scythe

The Rpn10 subunit of the 26S proteasome can bind to polyubiquitinoylated and/or ubiquitin‐like proteins via ubiquitin‐interacting motifs (UIMs). Vertebrate Rpn10 consists of five distinct spliced isoforms, but the specific functions of these variants remain largely unknown. We report here that one of the alternative products of Xenopus Rpn10, named Xrpn10c, functions as a specific receptor for Scythe/BAG‐6, which has been reported to regulate Reaper‐induced apoptosis. Deletional analyses revealed that Scythe has at least two distinct domains responsible for its binding to Xrpn10c. Conversely, an Xrpn10c has a UIM‐independent Scythe‐binding site. The forced expression of a Scythe mutant protein lacking Xrpn10c‐binding domains in Xenopus embryos induces inappropriate embryonic death, whereas the wild‐type Scythe did not show any abnormality. The results indicate that Xrpn10c‐binding sites of Scythe act as an essential segment linking the ubiquitin/proteasome machinery to the control of proper embryonic development.

Scythe regulates apoptosis through modulating ubiquitin-mediated proteolysis of the Xenopus elongation factor XEF1AO

Scythe was originally identified as a novel Reaper-binding anti-apoptotic protein, although the mechanisms of its functions remain largely obscure. Our previous analysis revealed that Scythe can bind to a proteasomal subunit via N-terminal domains and that the domains are required for appropriate development of Xenopus embryos. In the present study, we show evidence that the N-terminus of Scythe interacts with XEF1AO, a maternal form of Xenopus laevis EF1A that was suggested to be a potential inducer of apoptosis in vertebrates, and that the binding enhances the poly-ubiquitin modification and subsequent degradation of XEF1AO. Scythe is required for degradation of XEF1AO, since immunodepletion of Scythe from embryonic extracts stabilized XEF1AO significantly. Furthermore, we show that apoptosis induced by accumulation of XEF1AO can be suppressed by co-expression of the full-length form of Scythe. These observations indicate that the proteolytic regulation of XEF1AO, mediated through Scythe, is essential to prevent inappropriate accumulation of XEF1AO and resulting apoptotic events during the course of Xenopus development.

Involvement of Rel/NF‐κB in regulation of ascidian notochord formation

The Rel/NF‐κB family is known to be involved in a wide variety of biological processes, including morphogenesis. In the present study, two protochordate cDNA clones encoding Rel/NF‐κB proteins, named As‐rel1 and As‐rel2, were isolated from a fertilized egg cDNA library of the ascidian Halocynthia roretzi. The As‐rel1 protein is a typical Rel/NF‐κB family member, containing a Rel homology domain, a nuclear localization sequence and a C‐terminal putative transcription activation domain, while the As‐rel2 protein is a novel Rel/NF‐κB family member that lacks a nuclear localization sequence and the C‐terminal domain. Northern blot analyses showed that both transcripts were maternally expressed and that their expression changed during development of H. roretzi embryos. Although injection of the As‐rel2 mRNA into H. roretzi fertilized eggs had little effect on embryonic development, injection of the As‐rel1 mRNA interfered greatly with notochord formation, resulting in a shortened tail with a reduced number of notochord cells. In contrast, embryos co‐injected with As‐rel1 and As‐rel2 mRNA developed normally, indicating that the As‐rel2 protein rescued the defect in notochord formation induced by the injection of As‐rel1 mRNA alone. These results strongly suggest that the As‐rel1 protein functions as a suppressor in ascidian notochord formation, while the As‐rel2 protein has an antagonistic effect on the action of the As‐rel1 protein.

OMA-1 is a P granules-associated protein that is required for germline specification in Caenorhabditis elegans embryos.

In Caenorhabditis elegans, CCCH‐type zinc‐finger proteins have been shown to be involved in the differentiation of germ cells during embryonic development. Previously, we and others have identified novel redundant CCCH‐type zinc‐finger proteins, OMA‐1 and OMA‐2, that are involved in oocyte maturation. In this study, we report that the cytoplasmic expression level of OMA‐1 protein was largely reduced after fertilization. In contrast to its cytoplasmic degradation, OMA‐1 was found to accumulate exclusively on P granules in germline blastomeres during embryogenesis. A notable finding is that embryos with partially suppressed oma‐1; oma‐2 expression showed inappropriate germline specification, including abnormal distributions of PGL‐1, MEX‐1 and PIE‐1 proteins. Thus, our results suggest that oma gene products are novel multifunctional proteins that participate in crucial processes for germline specification during embryonic development.

A novel signaling pathway mediated by the nuclear targeting of C-terminal fragments of mammalian Patched 1.

Background Patched 1 (Ptc1) is a polytopic receptor protein that is essential for growth and differentiation. Its extracellular domains accept its ligand, Sonic Hedgehog, while the function of its C-terminal intracellular domain is largely obscure. Principal Findings In this study, we stably expressed human Ptc1 protein in HeLa cells and found that it is subjected to proteolytic cleavage at the C-terminus, resulting in the generation of soluble C-terminal fragments. These fragments accumulated in the nucleus, while the N-terminal region of Ptc1 remained in the cytoplasmic membrane fractions. Using an anti-Ptc1 C-terminal domain antibody, we provide conclusive evidence that C-terminal fragments of endogenous Ptc1 accumulate in the nucleus of C3H10T1/2 cells. Similar nuclear accumulation of endogenous C-terminal fragments was observed not only in C3H10T1/2 cells but also in mouse embryonic primary cells. Importantly, the C-terminal fragments of Ptc1 modulate transcriptional activity of Gli1. Conclusions Although Ptc1 protein was originally thought to be restricted to cell membrane fractions, our findings suggest that its C-terminal fragments can function as an alternative signal transducer that is directly transported to the cell nucleus.

The 26S proteasome Rpn10 gene encoding splicing isoforms: Evolutional conservation of the genomic organization in vertebrates

Abstract Recognition of polyubiquitinated substrates by the 26S proteasome is a key step in the selective degradation of various cellular proteins. The Rpn10 subunit of the 26S proteasome can bind polyubiquitin conjugates in vitro.We have previously reported the unique diversity of Rpn10, which differs from other multiple proteasome subunits, and that the mouse Rpn10 mRNA family is generated from a single gene by developmentally regulated alternative splicing. To determine whether such alternative splicing mechanisms occur in other species, we searched for Rpn10 isoforms in databases and in our original PCR products. Here we report the genomic organization of the Rpn10 gene in lower vertebrates and provide evidence for the competent generation of distinct forms of Rpn10 by alternative splicing through evolution.

cDNA cloning and characterization of an osmotically sensitive TRP channel from ascidian eggs

The transient receptor potential (TRP) ion channels are thought to be involved in the entry of calcium ion into cells. In this study, we isolated a cDNA clone, HrTRPV, that shows high homology to Caenorhabditis elegans OSM-9, a TRPV subfamily member of the TRP family, from a Halocynthia roretzi fertilized egg cDNA library. We analyzed its properties using HrTRPV-transfected cells. Upon reduction of extracellular osmolarity, the intracellular calcium concentration was found to increase in HrTRPV-transfected cells. This increase in intracellular calcium concentration was dependent on the presence of extracellular calcium ion and was inhibited by treatment with gadolinium ion, a stretch-activated calcium channel blocker. Thus, these results indicate that ascidian egg HrTRPV is an osmotically sensitive TRP channel.

A conserved island of BAG6/Scythe is related to ubiquitin domains and participates in short hydrophobicity recognition

BAG6 (also called Scythe) interacts with the exposed hydrophobic regions of newly synthesized proteins and escorts them to the degradation machinery through mechanisms that remain to be elucidated. In this study, we provide evidence that BAG6 physically interacts with the model defective protein substrate CL1 in a manner that depends directly on its short hydrophobicity. We found that the N terminus of BAG6 contains an evolutionarily conserved island tentatively designated the BAG6 ubiquitin‐linked domain. Partial deletion of this domain in the BAG6 N‐terminal fragment abolished in cell recognition of polyubiquitinated polypeptides as well as the hydrophobicity‐mediated recognition of the CL1 degron in cell and in vitro. These observations suggest a mechanism whereby the BAG6 ubiquitin‐linked domain provides a platform for discriminating substrates with shorter hydrophobicity stretches as a signal for defective proteins.

Knocking out ubiquitin proteasome system function in vivo and in vitro with genetically encodable tandem ubiquitin.

At present, the 26S proteasome-specific inhibitor is not available. We constructed polyubiquitin derivatives that contained a tandem repeat of ubiquitins and were insensitive to ubiquitin hydrolases. When these artificial polyubiquitins (tUbs, tandem ubiquitins) were overproduced in the wild-type yeast strain, growth was strongly inhibited, probably because of inhibition of the 26S proteasome. We also found that several substrates of the ubiquitin-proteasome pathway were stabilized by expressing tUbs in vivo. tUbs containing four units or more of the ubiquitin monomer were found to form a complex with the 26S proteasome. We showed that tUb bound to the 26S proteasome inhibited the in vitro degradation of polyubiquitinylated Sic1 by the 26S proteasome. When tUB6 (six-mer) messenger RNA was injected into Xenopus embryos, cell division was inhibited, suggesting that tUb can be used as a versatile inhibitor of the 26S proteasome.