Characterization of human junctophilin subtype genes

Junctophilin (JP) subtypes, namely JP-1, 2, and 3, have been currently identified in excitable cells and constitute a novel family of junctional membrane complex proteins. Our studies have suggested that JPs take part in the formation of junctional membrane complexes by spanning the membrane of the intracellular Ca(2+) store and interacting with the cell-surface membrane. In this report we describe the primary structures, genomic organization, and tissue distribution of human JP subtypes. By cloning and analyzing human genomic DNA segments, the protein-coding sequence interrupted with four introns was defined in each JP gene. The deduced human JP subtypes shared characteristic structural features with their rabbit and mouse counterparts. Genomic mapping demonstrated that JP genes do not cluster on the human genome. RNA blot hybridization indicated that tissue-specific expression patterns of JP genes in human are essentially the same as those in mouse; skeletal muscle contained both JP-1 and JP-2 mRNAs, the heart predominantly expressed JP-2 mRNA, and the brain specifically contained JP-3 mRNA. In the light of this, we propose intramolecular domains of JP subtypes based on the structural and functional characteristics.     

A Naturally Occurring Canine Model of Autosomal Recessive Congenital Stationary Night Blindness

Congenital stationary night blindness (CSNB) is a non-progressive, clinically and genetically heterogeneous disease of impaired night vision. We report a naturally-occurring, stationary, autosomal recessive phenotype in beagle dogs with normal daylight vision but absent night vision. Affected dogs had normal retinas on clinical examination, but showed no detectable rod responses. They had “negative-type” mixed rod and cone responses in full-field ERGs. Their photopic long-flash ERGs had normal OFF-responses associated with severely reduced ON-responses. The phenotype is similar to the Schubert-Bornschein form of complete CSNB in humans. Homozygosity mapping ruled out most known CSNB candidates as well as CACNA2D4 and GNB3. Three remaining genes were excluded based on sequencing the open reading frame and intron-exon boundaries (RHO, NYX), causal to a different form of CSNB (RHO) or X-chromosome (NYX, CACNA1F) location. Among the genes expressed in the photoreceptors and their synaptic terminals, and mGluR6 cascade and modulators, reduced expression of GNAT1, CACNA2D4 and NYX was observed by qRT-PCR in both carrier (n = 2) and affected (n = 2) retinas whereas CACNA1F was down-regulated only in the affecteds. Retinal morphology revealed normal cellular layers and structure, and electron microscopy showed normal rod spherules and synaptic ribbons. No difference from normal was observed by immunohistochemistry (IHC) for antibodies labeling rods, cones and their presynaptic terminals. None of the retinas showed any sign of stress. Selected proteins of mGluR6 cascade and its modulators were examined by IHC and showed that PKCα weakly labeled the rod bipolar somata in the affected, but intensely labeled axonal terminals that appeared thickened and irregular. Dendritic terminals of ON-bipolar cells showed increased Goα labeling. Both PKCα and Goα labeled the more prominent bipolar dendrites that extended into the OPL in affected but not normal retinas. Interestingly, RGS11 showed no labeling in the affected retina. Our results indicate involvement of a yet unknown gene in this canine model of complete CSNB.     

Small RNA class transition from siRNA/piRNA to miRNA during pre-implantation mouse development

Recent studies showed that small interfering RNAs (siRNAs) and Piwi-interacting RNA (piRNA) in mammalian germ cells play important roles in retrotransposon silencing and gametogenesis. However, subsequent contribution of those small RNAs to early mammalian development remains poorly understood. We investigated the expression profiles of small RNAs in mouse metaphase II oocytes, 8–16-cell stage embryos, blastocysts and the pluripotent inner cell mass (ICM) using high-throughput pyrosequencing. Here, we show that during pre-implantation development a major small RNA class changes from retrotransposon-derived small RNAs containing siRNAs and piRNAs to zygotically synthesized microRNAs (miRNAs). Some siRNAs and piRNAs are transiently upregulated and directed against specific retrotransposon classes. We also identified miRNAs expression profiles characteristic of the ICM and trophectoderm (TE) cells. Taken together, our current study reveals a major reprogramming of functional small RNAs during early mouse development from oocyte to blastocyst.     

Pdk1 kinase regulates basal disease resistance through the OsOxi1-OsPti1a phosphorylation cascade in rice

The AGC kinase OsOxi1, which has been isolated as an interactor with OsPti1a, positively regulates basal disease resistance in rice. In eukaryotes, AGC kinase family proteins are regulated by 3-phosphoinositide-dependent protein kinase 1 (Pdk1). In Arabidopsis, AtPdk1 directly interacts with phosphatidic acid, which functions as a second messenger in both biotic and abiotic stress responses. However, the functions of Pdk1 are poorly understood in plants. We show here that OsPdk1 acts upstream of the OsOxi1-OsPti1a signal cascade in disease resistance in rice. OsPdk1 interacts with OsOxi1 and phosphorylates the Ser283 residue of OsOxi1 in vitro. To investigate whether OsPdk1 is involved in immunity that is triggered by microbial-associated molecular patterns, we analyzed the phosphorylation status of OsPdk1 in response to chitin elicitor. Like OsOxi1, OsPdk1 is rapidly phosphorylated in response to chitin elicitor, suggesting that OsPdk1 participates in signal transduction through pathogen recognition. The overexpression of OsPdk1 enhanced basal resistance against a blast fungus, Magnaporthe oryzae, and a bacterial pathogen, Xanthomonas oryzae pv. oryzae (Xoo). Taken together, these results suggest that OsPdk1 positively regulates basal disease resistance through the OsOxi1-OsPti1a phosphorylation cascade in rice.     

Unique features of the rice blast resistance <Emphasis Type="Italic">Pish</Emphasis> locus revealed by large scale retrotransposon-tagging

Background  

R gene-mediated resistance is one of the most effective mechanisms of immunity against pathogens in plants. To date some components that regulate the primary steps of plant immunity have been isolated, however, the molecular dissection of defense signaling downstream of the R proteins remains to be completed. In addition, R genes are known to be highly variable, however, the molecular mechanisms responsible for this variability remain obscure.     

Differential expression of protein phosphatase type 1 isotypes and nucleolin during cell cycle arrest

In the present study, we examined the expression and cytolocalization of protein phosphatase type 1 (PP1) isoforms and nucleolin in human osteoblastic cell line MG63 cells at two boundaries in the cell cycle. We treated MG63 cells with hydroxyurea and nocodazole to arrest the cells at the G(1)/S and G(2)/M boundaries, respectively. As judged from the results of Western blot analysis, PP1 isoforms were expressed differently at each boundary of the cell cycle. Nucleolin was also shown to have a different expression pattern at each boundary. In the hydroxyurea-treated cells, nucleolus-like bodies were bigger in size and decreased in number compared with those in asynchronized cells. However, the subcellular localization of PP1s and nucleolin was not changed. Anti-nucleolin antibody interacted with 110-kDa and 95-kDa proteins present in asynchronized cells and in the cells treated with hydroxyurea. Treatment of the cells with nocodazole decreased the level of the 95-kDa form of nucleolin. In the nocodazole-treated cells, it was impossible to distinguish the distribution of each protein. The phosphorylation status of nucleolin in the cell cycle arrested samples was examined by 2D-IEF-PAGE followed by Western blot analysis. In the case of asynchronized cells or hydroxyurea-treated ones, nucleolin was located at a basic isoelectric point (dephosphorylated status); whereas in the G(2)/M arrest cells, the isoelectric point of nucleolin shifted to an acidic status, indicating that nucleolin was phosphorylated. The present results indicate that PP1 and nucleolin were differently expressed at G(1)/S and G(2)/M boundaries of the cell cycle and acted in a different fashion during cell-cycle progression.     

In vitro and in silico analysis reveals an efficient algorithm to predict the splicing consequences of mutations at the 5' splice sites

We have found that two previously reported exonic mutations in the PINK1 and PARK7 genes affect pre-mRNA splicing. To develop an algorithm to predict underestimated splicing consequences of exonic mutations at the 5′ splice site, we constructed and analyzed 31 minigenes carrying exonic splicing mutations and their derivatives. We also examined 189 249 U2-dependent 5′ splice sites of the entire human genome and found that a new variable, the SD-Score, which represents a common logarithm of the frequency of a specific 5′ splice site, efficiently predicts the splicing consequences of these minigenes. We also employed the information contents (R_i) to improve the prediction accuracy. We validated our algorithm by analyzing 32 additional minigenes as well as 179 previously reported splicing mutations. The SD-Score algorithm predicted aberrant splicings in 198 of 204 sites (sensitivity = 97.1%) and normal splicings in 36 of 38 sites (specificity = 94.7%). Simulation of all possible exonic mutations at positions −3, −2 and −1 of the 189 249 sites predicts that 37.8, 88.8 and 96.8% of these mutations would affect pre-mRNA splicing, respectively. We propose that the SD-Score algorithm is a practical tool to predict splicing consequences of mutations affecting the 5′ splice site.