p2 of rice stripe virus (RSV) interacts with OsSGS3 and is a silencing suppressor

A rice cDNA library was screened by a galactosidase 4 (Gal4)-based yeast two-hybrid system with Rice stripe virus (RSV) p2 as bait. The results revealed that RSV p2 interacted with a rice protein exhibiting a high degree of identity with Arabidopsis thaliana suppressor of gene silencing 3 (AtSGS3). The interaction was confirmed by bimolecular fluorescence complementation assay. SGS3 has been shown to be involved in sense transgene-induced RNA silencing and in the biogenesis of trans-acting small interfering RNAs (ta-siRNAs), possibly functioning as a cofactor of RNA-dependent RNA polymerase 6 (RDR6). Given the intimate relationships between virus and RNA silencing, further experiments were conducted to show that p2 was a silencing suppressor. In addition, p2 enhanced the accumulation and pathogenicity of Potato virus X in Nicotiana benthamiana. Five genes that have been demonstrated to be targets of TAS3-derived ta-siRNAs were up-regulated in RSV-infected rice. The implications of these findings are discussed.     

The Wilms' tumor suppressor Wt1 encodes a transcriptional activator of the class IV POU-domain factor Pou4f2 (Brn-3b)

The Wilms' tumor gene Wt1 encodes a zinc finger protein, which is required for normal formation of the genitourinary system and mesothelial tissues. Our recent findings indicate that Wt1 also plays a critical role in the development of ganglion cells in the vertebrate retina. Here we show that the POU-domain factor Pou4f2 (formerly Brn-3b), which is necessary for retinal ganglion cell survival, is up-regulated in human embryonic kidney (HEK)293 cells with stable Wt1 expression. Consistent with our previous observations of increased Pou4f2 mRNA in stably Wt1-transfeced HEK293 cells [EMBO J. 21 (2002) 1398], endogenous Pou4f2 was also elevated at the protein level in the HEK293 transfectants as well as in U2OS osteosarcoma cells that expressed an inducible Wt1 isoform. Transient co-transfection of a Wt1 expression construct activated a Pou4f2 promoter-reporter construct approximately 4-fold. Stimulation of the Pou4f2 promoter required a Wt1 binding element that was similar to a degenerative consensus site previously identified in other Wt1 responsive genes. Double-immunofluorescent labeling revealed co-expression of Pou4f2 and Wt1 in glomerular podocytes of adult kidney and in developing retinal ganglion cells of mouse embryos. Pou4f2 immunoreactivity was absent from the retinas of Wt1(-/-) embryos. In conclusion, we identified Pou4f2 as a novel downstream target gene of Wt1. Co-localization of both proteins in glomerular podocytes of the kidney and in developing retinal ganglion cells suggests a role for Wt1-Pou4f2 interaction in these tissues.     

The small GTPase rab5 functions as a regulatory factor in the early endocytic pathway.

We have investigated the in vivo functional role of rab5, a small GTPase associated with the plasma membrane and early endosomes. Wild-type rab5 or rab5-ile133, a mutant protein defective in GTP binding, was overexpressed in baby hamster kidney cells. In cells expressing the rab5ile 133 protein, the rate of endocytosis was decreased by 50% compared with normal, while the rate of recycling was not significantly affected. The morphology of early endosomes was also drastically changed by the mutant protein, which induced accumulation of small tubules and vesicles at the periphery of the cell. Surprisingly, overexpression of wild-type rab5 accelerated the uptake of endocytic markers and led to the appearance of atypically large early endosomes. We conclude that rab5 is a rate-limiting component of the machinery regulating the kinetics of membrane traffic in the early endocytic pathway.     

Cloning and expression of ARMC3_v2, a novel splicing variant of the human ARMC3 gene

ARM genes, whose polypeptide consist of Armadillo/beta-catenin-like repeats (ARM) domain(s), exist ubiquitously from fly to vertebrates. These genes have multiple functions in signal transduction, development, cell adhesion and mobility, tumor initiation and metastasis. In this study, we have isolated a novel splicing variant of ARMC3 from human fetal brain, which is 2439 bp, encoding a 688-amino acid polypeptide that contains three typical ARM domains. The cDNA called ARMC3_v2 and the original called ARMC3_v1 (GeneBank: BC039312) are both located on the human chromosome 10p12.23. RT-PCR analysis in our work showed that ARMC3_v2 was detected in human skeletal muscle, liver, spleen and thymus; in contrast, ARMC3_v1 in skeletal muscle, lung, prostate and testis.     

Human DDX3 functions in translation and interacts with the translation initiation factor eIF3

The conserved RNA helicase DDX3 is of major medical importance due to its involvement in numerous cancers, human hepatitis C virus (HCV) and HIV. Although DDX3 has been reported to have a wide variety of cellular functions, its precise role remains obscure. Here, we raised a new antibody to DDX3 and used it to show that DDX3 is evenly distributed throughout the cytoplasm at steady state. Consistent with this observation, HA-tagged DDX3 also localizes to the cytoplasm. RNAi of DDX3 in both human and Drosophila cells shows that DDX3 is required for cell viability. Moreover, using RNAi, we show that DDX3 is required for expression of protein from reporter constructs. In contrast, we did not detect a role for DDX3 in nuclear steps in gene expression. Further insight into the function of DDX3 came from the observation that its major interaction partner is the multi-component translation initiation factor eIF3. We conclude that a primary function for DDX3 is in protein translation, via an interaction with eIF3.