Junctophilins: a novel family of junctional membrane complex proteins

Junctional complexes between the plasma membrane (PM) and endoplasmic/sarcoplasmic reticulum (ER/ SR) are a common feature of all excitable cell types and mediate cross-talk between cell surface and intracellular ion channels. We have identified the junctophilins (JPs), a novel conserved family of proteins that are components of the junctional complexes. JPs are composed of a carboxy-terminal hydrophobic segment spanning the ER/SR membrane and a remaining cytoplasmic domain that shows specific affinity for the PM. In mouse, there are at least three JP subtypes: JP-1, -2, and -3. JP-2 is abundantly expressed in the heart, and mutant mice lacking JP-2 exhibited embryonic lethality. Cardiac myocytes from the mutant mice showed deficiency of the junctional membrane complexes and abnormal Ca2+ transients. Our results suggest that JPs are important components of junctional membrane complexes.     

Physiological and genetic analyses of aluminium tolerance in rice, focusing on root growth during germination

Aluminium (Al) ion limits root growth of plants in acidic soils, and rice exhibits the highest level of Al-tolerance among graminous crops. To elucidate Al-tolerance mechanisms in rice, response to Al was compared between rice (Oryza sativa L., cv. Nipponbare) and wheat (Triticum aestivum L., cv. ET8), focusing on seminal root growth at seedling stage and germination stage. At seedling stage, rice and wheat were similarly sensitive to Al in both dose- and time-dependent manner during a 24-h Al exposure. On the contrary, at germination stage, rice was more tolerant to Al than wheat, and wheat roots displayed the loss of plasma membrane integrity more extensively than rice. A rice mutant exhibiting Al hypersensitivity at germination stage was obtained by screening 42,840 R2 progeny derived from the regenerated R0 plants of Nipponbare and thereafter confirmation of the mutant phenotype in R3 progeny. At germination stage, root growth of the mutant was strongly inhibited in the presence of Al but not in the absence of Al. However, at seedling stage, root growth of the mutant and wild type was similarly tolerant to Al. Taken together, we conclude that rice possesses Al-tolerant function that is under genetic control and specifically operates for root growth at germination stage, making rice more tolerant to Al than wheat.     

Conservation of the E-function for Floral Organ Identity in Rice Revealed by the Analysis of Tissue Culture-induced Loss-of-Function Mutants of the <Emphasis Type="Italic">OsMADS1</Emphasis> Gene

Rapid progress in studies on flower development has resulted in refining the classical ‘ABC model’ into a new ‘ABCDE model’ to explain properly the regulation of floral organ identity. Conservation of E-function for flower organ identity among the dicotyledonous (dicot) plants has been revealed. However, its conservation in monocotyledonous (monocot) plants remains largely unknown. Here, we show the conservation of E-function in rice (Oryza sativaL.) by characterizing tissue culture-induced mutants of two MADS-box genes, OsMADS1and OsMADS5, which form a subclade within the well-supported clade of SEP-genes (E-function) phylogeny. Severe loss-of-function mutations of OsMADS1cause complete homeotic conversion of organs (lodicules, stamens, and carpels) of three inner whorls into lemma- and palea-like structures. Such basic deformed structure is reiterated along with the pedicel at the center of the same floret, indicating the loss of determinacy of the flower meristem. These phenotypes resemble the phenotypes caused by mutations of the dicot E-class genes, such as the Arabidopsis SEP123(SEPALLATA1/2/3) and the petunia FBP2(Floral Binding Protein 2), suggesting that OsMADS1play a very similar role in rice to that of defined E-class genes in dicot plants. In case of the loss-of-function mutation of OsMADS5, no defect in either panicles or vegetative organs was observed. These results demonstrate that OsMADS1clearly possesses E-function, and so, E-function is fundamentally conserved between dicot plants and rice, a monocot model plant.     

Differential regulation of <Emphasis Type="Italic">chlorophyll a oxygenase</Emphasis> genes in rice

Chlorophyll b is synthesized from chlorophyll a by chlorophyll a oxygenase. We have identified two genes (OsCAO1 and OsCAO2) from the rice genome that are highly homologous to previously studied chlorophyll a oxygenase (CAO) genes. They are positioned in tandem, probably resulting from recent gene duplications. The proteins they encode contain two conserved functional motifs – the Rieske Fe–sulfur coordinating center and a non-heme mononuclear Fe-binding site. OsCAO1 is induced by light and is preferentially expressed in photosynthetic tissues. Its mRNA level decreases when plants are grown in the dark. In contrast, OsCAO2 mRNA levels are higher under dark conditions, and its expression is down-regulated by exposure to light. To elucidate the physiological function of the CAO genes, we have isolated knockout mutant lines tagged by T-DNA or Tos17. Mutant plants containing a T-DNA insertion in the first intron of the OsCAO1 gene have pale green leaves, indicating chlorophyll b deficiency. We have also isolated a pale green mutant with a Tos17 insertion in that OsCAO1 gene. In contrast, OsCAO2 knockout mutant leaves do not differ significantly from the wild type. These results suggest that OsCAO1 plays a major role in chlorophyll b biosynthesis, and that OsCAO2 may function in the dark.     

Distinct and cooperative functions of phytochromes A, B, and C in the control of deetiolation and flowering in rice

We have isolated phytochrome B (phyB) and phyC mutants from rice (Oryza sativa) and have produced all combinations of double mutants. Seedlings of phyB and phyB phyC mutants exhibited a partial loss of sensitivity to continuous red light (Rc) but still showed significant deetiolation responses. The responses to Rc were completely canceled in phyA phyB double mutants. These results indicate that phyA and phyB act in a highly redundant manner to control deetiolation under Rc. Under continuous far-red light (FRc), phyA mutants showed partially impaired deetiolation, and phyA phyC double mutants showed no significant residual phytochrome responses, indicating that not only phyA but also phyC is involved in the photoperception of FRc in rice. Interestingly, the phyB phyC double mutant displayed clear R/FR reversibility in the pulse irradiation experiments, indicating that both phyA and phyB can mediate the low-fluence response for gene expression. Rice is a short-day plant, and we found that mutation in either phyB or phyC caused moderate early flowering under the long-day photoperiod, while monogenic phyA mutation had little effect on the flowering time. The phyA mutation, however, in combination with phyB or phyC mutation caused dramatic early flowering.     

Identification of a putative voltage-gated Ca2+ channel as a key regulator of elicitor-induced hypersensitive cell death and mitogen-activated protein kinase activation in rice

Elicitor-triggered transient membrane potential changes and Ca2+ influx through the plasma membrane are thought to be important during defense signaling in plants. However, the molecular bases for the Ca2+ influx and its regulation remain largely unknown. Here we tested effects of overexpression as well as retrotransposon (Tos17)-insertional mutagenesis of the rice two-pore channel 1 (OsTPC1), a putative voltage-gated Ca(2+)-permeable channel, on a proteinaceous fungal elicitor-induced defense responses in rice cells. The overexpressor showed enhanced sensitivity to the elicitor to induce oxidative burst, activation of a mitogen-activated protein kinase (MAPK), OsMPK2, as well as hypersensitive cell death. On the contrary, a series of defense responses including the cell death and activation of the MAPK were severely suppressed in the insertional mutant, which was complemented by overexpression of the wild-type gene. These results suggest that the putative Ca(2+)-permeable channel determines sensitivity to the elicitor and plays a role as a key regulator of elicitor-induced defense responses, activation of MAPK cascade and hypersensitive cell death.     

Ex vivo survival of peripheral blood mononuclear cells in sheep induced by bovine leukemia virus (BLV) mainly occurs in CD5- B cells that express BLV

Bovine leukemia virus (BLV) is the etiologic agent of enzootic bovine leukosis (EBL). In a previous report, we found that in a sheep model, only CD5(-) B cells proliferated clonally, while CD5(+) B cells rapidly decreased when the disease progressed to the lymphoma stage. We demonstrate here that, although both CD5(+) and CD5(-) B cells, but not CD4(+) T, CD8(+) T and gammadeltaTCR(+)T cells, are protected from spontaneous ex vivo apoptosis in sheep infected with wild-type and a mutant BLV that encodes a mutant Tax D247G protein with elevated trans-activation activity, only CD5(-) B cells become the main target for ex vivo survival when the disease proceeds to the persistent lymphocytotic stage, which showed an increased expansion of the CD5(-) B cells. In addition, we identified, by four-color flow cytometric analysis, that in CD5(-) B cells, the apoptotic rates of cells that expressed wild-type and mutant BLV were greatly decreased compared with those of BLV-negative cells. There was only a slight reduction in the apoptotic rates in BLV-positive cells from CD5(+) B cells. In addition, supernatants from peripheral blood mononuclear cell (PBMC) cultures from wild-type- and mutant BLV-infected sheep mainly protected CD5(-) B cells from spontaneous apoptosis. Our results suggest that, although BLV can protect both CD5(+) and CD5(-) B cells from ex vivo apoptosis, the mechanisms accounting for the ex vivo survival between these two B-cell subsets differ. Therefore, it appears that the phenotypic changes in cells that express CD5 at the lymphoma stage could result from a difference in susceptibility to apoptosis in CD5(+) and CD5(-) B cells in BLV-infected sheep.     

Chimeric RNA and 2'-O, 4'-C-ethylene-bridged nucleic acids have stronger activity than phosphorothioate oligodeoxynucleotides in induction of exon 19 skipping in dystrophin mRNA

Antisense phosphorothioate oligodeoxynucleotides against exon 19 of the dystrophin gene have been shown to induce exon 19 skipping and promote the expression of internally deleted dystrophin by correcting the translational reading frame. Because phosphorothioate oligonucleotides are associated with a variety of toxic nonantisense effects, several modifications of nucleic acid have been introduced to alleviate this toxicity. Recently, a 2'-O, 4'-C-ethylene-bridged nucleic acid (ENA trade mark, Sankyo Lifetech Co., Ltd., Tokyo, Japan) was reported to have high affinity to complementary RNA strands and be resistant to nuclease digestion. Here, we examined the ability of this modified nucleic acid to induce exon skipping. Oligonucleotides having the same sequence as the phosphorothioate oligonucleotides but with some stretches of modified backbone (2'-O-methyl RNA with an ENA5-mer at the 5'-end and 3'-end) (RNA/ENA chimera) were transfected into myocytes, and the expressed dystrophin mRNA was analyzed. The RNA/ENA chimera induced exon 19 skipping in a dose-dependent and time-dependent manner. Remarkably, the exon 19-skipping activity of the RNA/ENA chimera was more than 40 times stronger than that of the corresponding conventional phosphorothioate oligodeoxynucleotide. This is the first report of such strong activity of an RNA/ENA chimera in the induction of exon skipping in the dystrophin gene. This new technology will allow the development of less toxic antisense drugs, making long-term therapy possible.     

Double-stranded RNA mediates selective gene silencing of protein phosphatase type 1 delta isoform in HEK-293 cells

The reversible phosphorylation of proteins mediates cellular signals in eukaryotic cells. RNA interference inhibits the expression of genes and proteins in a sequence-specific manner and provides a tool to study the functions of target molecules. The effect of RNA interference on protein phosphatase isoforms in HEK-293 cells was examined. Protein phosphatase 1 delta (PP1delta) sequence-specific double-stranded RNA (dsRNA) inhibited mRNA and protein expression of the PP1delta. This RNA interference did not affect the expression of lphaand gamma1 isoforms of PP1. Transfection of antisense RNA specific for PP1delta also suppressed the expression of PP1delta. It was further demonstrated by an in vitro RNA cleavage assay that extracts of HEK-293 cells catalyzed the processing of dsRNA. This cell line had much stronger mRNA expression of Dicer, an RNase III-like enzyme, than did human osteoblastic MG63 cells. The present results show that RNA interference is a useful tool to distinguish between PP1 isoforms.     

Chromosome binding site of latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus is essential for persistent episome maintenance and is functionally replaced by histone H1

Latency-associated nuclear antigen 1 (LANA1) of Kaposi's sarcoma-associated herpesvirus (KSHV; human herpesvirus 8) persistently maintains a plasmid containing the KSHV latent origin of replication (oriP) as a closed circular episome in dividing cells. In this study, we investigated the involvement of chromosome binding activity of LANA1 in persistent episome maintenance. Deletion of the N-terminal 22 amino acids of LANA1 (DeltaN-LANA) inhibited the interaction with mitotic chromosomes in a human cell line, and the mutant concomitantly lost activity for the long-term episome maintenance of a plasmid containing viral oriP in a human B-cell line. However, a chimera of DeltaN-LANA with histone H1, a cellular chromosome component protein, rescued the association with mitotic chromosomes as well as the long-term episome maintenance of the oriP-containing plasmid. Our results suggest that tethering of KSHV episomes to mitotic chromosomes by LANA1 is crucial in mediating the long-term maintenance of viral episomes in dividing cells.