Yasuhiro Araki

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.

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.

Interaction between Ski7p and Upf1p is required for nonsense‐mediated 3′‐to‐5′ mRNA decay in yeast

Aberrant mRNAs containing premature termination codons (PTC‐mRNAs) are degraded by a conserved surveillance system, referred to as the nonsense‐mediated decay (NMD) pathway. Although NMD is reported to operate on the decapping and 5′‐to‐3′ exonucleolytic decay of PTC‐mRNAs without affecting deadenylation, a role for an opposite 3′‐to‐5′ decay pathway remains largely unexplored. In this study, we have characterized the 3′‐to‐5′ directed mRNA degradation in the yeast NMD pathway. PTC‐mRNAs are stabilized in yeast cells lacking the components of 3′‐to‐5′ mRNA‐decay machinery. The 3′‐to‐5′ directed degradation of PTC‐mRNAs proceeds more rapidly than that of the PTC‐free transcript, in a manner dependent on the cytoplasmic exosome and Upf proteins. Moreover, Upf1p, but not Upf2p, interacts physically with an N‐terminal domain of Ski7p, although the interaction requires Upf2p. The efficiency of 3′‐to‐5′ directed degradation of PTC‐mRNAs is impaired by overexpression of Ski7p N‐domain fragments that contain a sequence of the Upf1p‐interaction region. These data suggest that the activation of 3′‐to‐5′ directed NMD is mediated through the interaction between Upf1p and the Ski7p N domain.

Di-Ras: A distinct subgroup of Ras-family GTPases with unique biochemical properties

The small GTPase Ras family regulates a variety of cell functions including proliferation and differentiation. Here we have identified novel Ras members, human Di-Ras1 and Di-Ras2, belonging to a distinct branch of the GTPase family. Di-Ras1 and Di-Ras2 specifically expressed in heart and brain share 30–40% overall identity with other members of Ras family, however, they have the following characteristic substitutions at highly conserved regions among the Ras family. 1) Thr-63 and Ser-65 in Di-Ras are substituted for Ala-59 and Gln-61 positions in Ha-Ras, respectively, that are known to be critical for GTP hydrolysis. 2) Within the effector domains, Di-Ras has Ile at a position corresponding to Asp-33 in Ha-Ras, which is important for its interaction with the downstream effector Raf. As predicted by these substitutions, Di-Ras has only a quite low level of GTPase activity and exists predominantly as a GTP-bound form upon its expression in living cells. Moreover, Di-Ras fails to interact with the Ras-binding domain of Raf, resulting in no stimulation of mitogen-activated protein kinase. Interestingly, introduction of Di-Ras into HEK293T cells induces large cellular vacuolation. These findings raise the possibility that Di-Ras might regulate cell morphogenesis in a manner distinct from other members of Ras family.

Upf1 potentially serves as a RING-related E3 ubiquitin ligase via its association with Upf3 in yeast.

Three Upf proteins are essential to the nonsense-mediated mRNA decay (NMD) pathway. Although these proteins assemble on polysomes for recognition of aberrant mRNAs containing premature termination codons, the significance of this assembly remains to be elucidated. The Cys- and His-rich repeated N terminus (CH domain) of Upf1 has been implicated in its binding to Upf2. Here, we show that CH domain also plays a RING-related role for Upf1 to exhibit E3 ubiquitin ligase activity in yeast. Despite the sequence divergence from typical E3-RING fingers, the CH domain of yeast Upf1 specifically and directly interacted with the yeast E2 Ubc3. Interestingly, Upf1 served as a substrate for the in vitro self-ubiquitination, and the modification required its association with Upf3 rather than Upf2. Substitution of the coordinated Cys and His residues in the CH domain impaired not only self-ubiquitination of Upf1 but also rapid decay of aberrant mRNAs. These results suggest that Upf1 may serve as an E3 ubiquitin ligase upon its association with Upf3 and play an important role in signaling to the NMD pathway.

Caf1 regulates translocation of ribonucleotide reductase by releasing nucleoplasmic Spd1–Suc22 assembly

Appropriate supply of deoxyribonucleotides by the ribonucleotide reductase (RNR) complex is essential for DNA replication and repair. One recent model for the RNR activation in Schizosaccharomyces pombe is translocation of the regulatory subunit Suc22 from the nucleoplasm to the cytoplasm. The RNR inhibitory protein Spd1, which retains Suc22 in the nucleoplasm, is rapidly degraded upon DNA-replication stress, resulting in release of Suc22 to form the active RNR complex in the cytoplasm. Here, we show that Caf1, a component of the Ccr4–Not complex, is responsible for resistance of the replication stress and control of the Suc22 translocation. Caf1 is required not only for the stress-induced translocation of Suc22 from nucleoplasm to cytoplasm but also for the degradation of nucleoplasmic Spd1. DNA-replication stress appears to allow Caf1 to interact with Suc22, resulting in release of the nucleoplasmic Spd1–Suc22 assembly. Taken together, these results suggest a novel function of Caf1 as a key regulator in the stress-induced RNR activation.

NGN/IMS-Based Ubiquitous Health Monitoring System

In this demonstration we present an ubiquitous health monitoring system based on the NGN/IMS (Next Generation Network/IP Multimedia Subsystem). It enables us to be taken care of by medical professionals anytime, anywhere using portable medical sensors and mobile networks. To provide seamless health monitoring services, an open, secure and functional platform is required. Such a platform should support multiple functions such as real-time transfer, event notification, and continuous data accumulation. The NGN/IMS is a potential platform to fulfill these requirements, because it is a standard-based open platform, which provides AAA (Authentication, Authorization and Accounting), QoS (Quality of Service) support, event notification and a data management server. We present a system design and a prototype implementation on a NGN/IMS testbed.

Design and Implementation of Sensor Network on the NGN/IMS

The sensor networks had been used for various application areas and showed effectiveness under the prior application planning and right circumstances that includes cost, data handling and other resources. In order to generalize, it is required to reduce initial cost and use data effectively. This paper proposes connecting a sensor network with the IMS that is the service platform of the NGN to solve these requirements. This paper shows prototype implementation of proposed system for collecting electric power consumption.

Purification and Analysis of RIN Family‐Novel Rab5 GEFs

The small GTPase Rab5 plays important roles in membrane budding and trafficking in the early endocytic pathways, and the activation of this GTPase is mediated by several guanine nucleotide exchange factors (GEFs) at each of the transport steps. The RIN family has been identified as GEFs for Rab5 and shown to possess unique biochemical properties. The RIN family preferentially interacts with an activated form of Rab5, although it enhances guanine nucleotide exchange reaction. Moreover, biochemical analysis indicates that the RIN family functions as a tetramer. In this chapter, we describe the isolation of the recombinant RIN family via expression in Spodoptera frugiperda (Sf9) insect cells and in mammalian cells. In addition, functional analysis is also provided to assess the physiological properties of the RIN family.

NGN/IMS-based ubiquitous health monitoring system — Data format design

Due to people!s concern for a healthy life, there is a rising need for a health monitoring available anytime and anywhere. We present an ubiquitous health monitoring system based on the NGN/IMS(Next Generation Network/IP Multimedia Subsystem) and proper data format design for ubiquitous health monitoring system.