Toshiaki Katada

The eukaryotic polypeptide chain releasing factor (eRF3/GSPT) carrying the translation termination signal to the 3'-poly(A) tail of mRNA-Direct association of eRF3/GSPT with polyadenylate-binding protein

The mammalian GTP-binding protein GSPT, whose carboxyl-terminal sequence is homologous to the eukaryotic elongation factor EF1α, binds to the polypeptide chain releasing factor eRF1 to function as eRF3 in the translation termination. The amino-terminal domain of GSPT was, however, not required for the binding. Search for other GSPT-binding proteins in yeast two-hybrid screening system resulted in the identification of a cDNA encoding polyadenylate-binding protein (PABP), whose amino terminus is associating with the poly(A) tail of mRNAs presumably for their stabilization. The interaction appeared to be mediated through the carboxyl-terminal domain of PABP and the amino-terminal region of GSPT. Interestingly, multimerization of PABP with poly(A), which is ascribed to the action of its carboxyl-terminal domain, was completely inhibited by the interaction with the amino-terminal domain of GSPT. These results indicate that GSPT/eRF3 may play important roles not only in the termination of protein synthesis but also in the regulation of mRNA stability. Thus, the present study is the first report showing that GSPT/eRF3 carries the translation termination signal to 3′-poly(A) tail ubiquitously present in eukaryotic mRNAs.

Ski7p G protein interacts with the exosome and the Ski complex for 3′-to-5′ mRNA decay in yeast

Two cytoplasmic mRNA‐decay pathways have been characterized in yeast, and both are initiated by shortening of the 3′‐poly(A) tail. In the major 5′‐to‐3′ decay pathway, the deadenylation triggers removal of the 5′‐cap, exposing the transcript body for 5′‐to‐3′ degradation. An alternative 3′‐to‐5′ decay pathway also follows the deadenylation and requires two multi‐complexes: the exosome containing various 3′‐exonucleases and the Ski complex consisting of the RNA helicase Ski2p, Ski3p and Ski8p. In addition, Ski7p, which has an N‐terminal domain and a C‐terminal elongation factor 1α‐like GTP‐binding domain, is involved in the 3′‐to‐5′ decay. However, physical interaction between the exosome and the Ski complex, together with the function of Ski7p, has remained unknown. Here we report that the N domain of Ski7p is required and sufficient for the 3′‐to‐5′ decay. Furthermore, the exosome and the Ski complex interact with the different regions of Ski7p N domain, and both interactions are required for the 3′‐to‐5′ decay. Thus, Ski7p G protein appears to function as a signal‐coupling factor between the two multi‐complexes operating in the 3′‐to‐5′ mRNA‐decay pathway.

Possible association of BLM in decreasing DNA double strand breaks during DNA replication

Blooms syndrome (BS) is a rare genetic disorder and the cells from BS patients show genomic instability and an increased level of sister chromatid exchange (SCE). We generated BLM−/− and BLM−/−/RAD54−/− DT40 cells from the chicken B‐lymphocyte line DT40. The BLM−/− DT40 cells showed higher sensitivity to methyl methanesulfonate and elevated levels of SCE as expected. The targeted integration frequency was also increased remarkably in BLM−/− cells. The SCE frequency increase in BLM−/− cells was considerably reduced and the enhanced targeted integration observed in BLM−/− cells was almost completely abolished in BLM−/−/RAD54−/− cells, indicating that a large portion of the SCE in BLM−/− cells occurs via homologous recombination, and homologous recombination events increase with the defect of BLM function. The BLM−/−/RAD54−/− cells showed a slow growth phenotype and an increased incidence of chromosome‐type breaks/gaps while each single mutant showed relatively small numbers of chromosome‐type breaks/gaps.

Translation termination factor eRF3 mediates mRNA decay through the regulation of deadenylation

Messenger RNA decay, which is a regulated process intimately linked to translation, begins with the deadenylation of the poly(A) tail at the 3′ end. However, the precise mechanism triggering the first step of mRNA decay and its relationship to translation have not been elucidated. Here, we show that the translation termination factor eRF3 mediates mRNA deadenylation and decay in the yeast Saccharomyces cerevisiae. The N-domain of eRF3, which is not necessarily required for translation termination, interacts with the poly(A)-binding protein PABP. When this interaction is blocked by means of deletion or overexpression of the N-domain of eRF3, half-lives of all mRNAs are prolonged. The eRF3 mutant lacking the N-domain is deficient in the poly(A) shortening. Furthermore, the eRF3-mediated mRNA decay requires translation to proceed, especially ribosomal transition through the termination codon. These results indicate that the N-domain of eRF3 mediates mRNA decay by regulating deadenylation in a manner coupled to translation.

MKK7 couples stress signalling to G2/M cell-cycle progression and cellular senescence

During the development of multicellular organisms, concerted actions of molecular signalling networks determine whether cells undergo proliferation, differentiation, death or ageing. Here we show that genetic inactivation of the stress signalling kinase, MKK7, a direct activator of JNKs in mice, results in embryonic lethality and impaired proliferation of hepatocytes. Beginning at passage 4–5, mkk7−/− mouse embryonic fibroblasts (MEFs) display impaired proliferation, premature senescence and G2/M cell cycle arrest. Similarly, loss of c-Jun or expression of a c-JunAA mutant in which the JNK phosphorylation sites were replaced with alanine results in a G2/M cell-cycle block. The G2/M cell-cycle kinase CDC2 was identified as a target for the MKK7–JNK–c-Jun pathway. These data show that the MKK7–JNK–c-Jun signalling pathway couples developmental and environmental cues to CDC2 expression, G2/M cell cycle progression and cellular senescence in fibroblasts.

A systematic genome-wide screen for mutations affecting organogenesis in Medaka, Oryzias latipes.

A large-scale mutagenesis screen was performed in Medaka to identify genes acting in diverse developmental processes. Mutations were identified in homozygous F3 progeny derived from ENU-treated founder males. In addition to the morphological inspection of live embryos, other approaches were used to detect abnormalities in organogenesis and in specific cellular processes, including germ cell migration, nerve tract formation, sensory organ differentiation and DNA repair. Among 2031 embryonic lethal mutations identified, 312 causing defects in organogenesis were selected for further analyses. From these, 126 mutations were characterized genetically and assigned to 105 genes. The similarity of the development of Medaka and zebrafish facilitated the comparison of mutant phenotypes, which indicated that many mutations in Medaka cause unique phenotypes so far unrecorded in zebrafish. Even when mutations of the two fish species cause a similar phenotype such as one-eyed-pinhead or parachute, more genes were found in Medaka than in zebrafish that produced the same phenotype when mutated. These observations suggest that many Medaka mutants represent new genes and, therefore, are important complements to the collection of zebrafish mutants that have proven so valuable for exploring genomic function in development.

RIN3: a novel Rab5 GEF interacting with amphiphysin II involved in the early endocytic pathway

The small GTPase Rab5, which cycles between active (GTP-bound) and inactive (GDP-bound) states, plays essential roles in membrane budding and trafficking in the early endocytic pathway. However, the molecular mechanisms underlying the Rab5-regulated processes are not fully understood other than the targeting event to early endosomes. Here, we report a novel Rab5-binding protein, RIN3, that contains many functional domains shared with other RIN members and additional Pro-rich domains. RIN3 displays the same biochemical properties as RIN2, the stimulator and stabilizer of GTP-Rab5. In addition, RIN3 exhibits its unique intracellular localization. RIN3 expressed in HeLa cells localized to cytoplasmic vesicles and the RIN3-positive vesicles contained Rab5 but not the early endosomal marker EEA1. Transferrin appeared to be transported partly through the RIN3-positive vesicles to early endosomes. RIN3 was also capable of interacting via its Pro-rich domain with amphiphysin II, which contains SH3 domain and participates in receptor-mediated endocytosis. Interestingly, cytoplasmic amphiphysin II was translocated into the RIN3- and Rab5-positive vesicles when co-expressed with RIN3. These results indicate that RIN3 biochemically characterized as the stimulator and stabilizer for GTP-Rab5 plays an important role in the transport pathway from plasma membrane to early endosomes.

cTAGE5 mediates collagen secretion through interaction with TANGO1 at endoplasmic reticulum exit sites

The mechanism of collagen secretion is not completely understood. It is found that cTAGE5 binds to TANGO1, and it is suggested that collagen VII export from the ER is driven by a cTAGE5/TANGO1 complex.

Identification, Sequence, and Expression of an Invertebrate Caveolin Gene Family from the Nematode Caenorhabditis elegans IMPLICATIONS FOR THE MOLECULAR EVOLUTION OF MAMMALIAN CAVEOLIN GENES

Caveolae are vesicular organelles that represent an appendage of the plasma membrane. Caveolin, a 21-24-kDa integral membrane protein, is a principal component of caveolae membranes in vivo. Caveolin has been proposed to function as a plasma membrane scaffolding protein to organize and concentrate signaling molecules within caveolae, including heterotrimeric G proteins (α and βγ subunits). In this regard, caveolin interacts directly with Gα subunits and can functionally regulate their activity. To date, three cDNAs encoding four subtypes of caveolin have been described in vertebrates. However, evidence for the existence of caveolin proteins in less complex organisms has been lacking. Here, we report the identification, cDNA sequence and genomic organization of the first invertebrate caveolin gene, Cavce (for caveolin from Caenorhabditis elegans). The Cavce gene, located on chromosome IV, consists of two exons interrupted by a 125-nucleotide intron sequence. The region of Cavce that is strictly homologous to mammalian caveolins is encoded by a single exon in Cavce. This suggests that mammalian caveolins may have evolved from the second exon of Cavce. Cavce is roughly equally related to all three known mammalian caveolins and, thus, could represent a common ancestor. Remarkably, the invertebrate Cavce protein behaves like mammalian caveolins: (i) Cavce forms a high molecular mass oligomer, (ii) assumes a cytoplasmic membrane orientation, and (iii) interacts with G proteins. A 20-residue peptide encoding the predicted G protein binding region of Cavce possesses “GDP dissociation inhibitor-like activity” with the same potency as described earlier for mammalian caveolin-1. Thus, caveolin appears to be structurally and functionally conserved from worms to man. In addition, we find that there are at least two caveolin-related genes expressed in C. elegans, defining an invertebrate caveolin gene family. These results establish the nematode C. elegans as an invertebrate model system to study caveolae and caveolin in vivo.

Role of activation of PIP5Kγ661 by AP‐2 complex in synaptic vesicle endocytosis

Synaptic vesicles (SVs) are retrieved by clathrin‐mediated endocytosis at the nerve terminals. Phosphatidylinositol 4,5‐bisphosphate [PI(4,5)P2] drives this event by recruiting the components of the endocytic machinery. However, the molecular mechanisms that result in local generation of PI(4,5)P2 remain unclear. We demonstrate here that AP‐2 complex directly interacts with phosphatidylinositol 4‐phosphate 5‐kinase γ661 (PIP5Kγ661), the major PI(4,5)P2‐producing enzyme in the brain. The β2 subunit of AP‐2 was found to bind to the C‐terminal tail of PIP5Kγ661 and cause PIP5Kγ661 activation. The interaction is regulated by PIP5Kγ661 dephosphorylation, which is triggered by depolarization in mouse hippocampal neurons. Finally, overexpression of the PIP5Kγ661 C‐terminal region in hippocampal neurons suppresses depolarization‐dependent SV endocytosis. These findings provide evidence for the molecular mechanism through which PIP5Kγ661 locally generates PI(4,5)P2 in hippocampal neurons and suggest a model in which the interaction trigger SV endocytosis.