Talin1 is required for cardiac Z-disk stabilization and endothelial integrity in zebrafish.

Talin (tln) binds and activates integrins to couple extracellular matrix-bound integrins to the cytoskeleton; however, its role in heart development is not well characterized. We identified the defective gene and the resulting cardiovascular phenotypes in zebrafish tln1(fl02k) mutants. The ethylnitrosourea-induced fl02k mutant showed heart failure, brain hemorrhage, and diminished cardiac and vessel lumens at 52 h post fertilization. Positional cloning revealed a nonsense mutation of tln1 in this mutant. tln1, but neither tln2 nor -2a, was dominantly expressed in the heart and vessels. Unlike tln1 and -2 in the mouse heart, the unique tln1 expression in the heart enabled us, for the first time, to determine the critical roles of Tln1 in the maintenance of cardiac sarcomeric Z-disks and endothelial/endocardial cell integrity, partly through regulating F-actin networks in zebrafish. The similar expression profiles of tln1 and integrin ß1b (itgb1b) and synergistic function of the 2 genes revealed that itgb1b is a potential partner for tln1 in the stabilization of cardiac Z-disks and vessel lumens. Taken together, the results of this work suggest that Tln1-mediated Itgß1b plays a crucial role in maintaining cardiac sarcomeric Z-disks and endothelial/endocardial cell integrity in zebrafish and may also help to gain molecular insights into congenital heart diseases.

Identification and expression analysis of the interferon-induced protein with tetratricopeptide repeats 5 (IFIT5) gene in duck (Anas platyrhynchos domesticus).

The interferon-induced proteins with tetratricopeptide repeats (IFITs) protein family mediates antiviral effects by inhibiting translation initiation, cell proliferation, and migration in the interferon (IFN) dependent innate immune system. Several members of this family, including IFIT1, IFIT2, IFIT3 and IFIT5, have been heavily studied in mammals. Avian species contain only one family member, IFIT5, and little is known about the role of this protein in birds. In this study, duck IFIT5 (duIFIT5) full-length mRNA was cloned by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of the cDNA ends (RACE). Based on the sequence obtained, we performed a series of bioinformatics analyses, and found that duIFIT5 was most similar to homologs in other avian species. Also, duIFIT5 contained eight conserved TPR motifs and two conserved multi-domains (TPR_11 and TPR_12). Finally, we used duck hepatitis virus type 1 (DHV-1) and polyriboinosinicpolyribocytidylic acid (poly (I:C)) as a pathogen or a pathogen-associated molecular pattern induction to infect three-day-old domestic ducklings. The liver and spleen were collected to detect the change in duIFIT5 transcript level upon infection by quantitative real-time PCR (qRT-PCR). DuIFIT5 expression rapidly increased after DHV-1 infection and maintained a high level, while the transcripts of duIFIT5 peaked at 8h after poly (I:C) infection and then returned to normal. Taken together, these results provide a greater understanding of avian IFIT5.

Genetic interaction between pku300 and fbn2b controls endocardial cell proliferation and valve development in zebrafish.

Abnormal cardiac valve morphogenesis is a common cause of human congenital heart disease. The molecular mechanisms regulating endocardial cell proliferation and differentiation into cardiac valves remain largely unknown, although great progress has been made on the endocardial contribution to the atrioventricular cushion and valve formation. We found that scotch tape(te382) (sco(te382)) encodes a novel transmembrane protein that is crucial for endocardial cell proliferation and heart valve development. The zebrafish sco(te382) mutant showed diminished endocardial cell proliferation, lack of heart valve leaflets and abnormal common cardinal and caudal veins. Positional cloning revealed a C946T nonsense mutation of a novel gene pku300 in the sco(te382) locus, which encoded a 540-amino-acid protein on cell membranes with one putative transmembrane domain and three IgG domains. A known G3935T missense mutation of fbn2b was also found ∼570 kb away from pku300 in sco(te382) mutants. The genetic mutant sco(pku300), derived from sco(te382), only had the C946T mutation of pku300 and showed reduced numbers of atrial endocardial cells and an abnormal common cardinal vein. Morpholino knockdown of fbn2b led to fewer atrial endocardial cells and an abnormal caudal vein. Knockdown of both pku300 and fbn2b phenocopied these phenotypes in sco(te382) genetic mutants. pku300 transgenic expression in endocardial and endothelial cells, but not myocardial cells, partially rescued the atrial endocardial defects in sco(te382) mutants. Mechanistically, pku300 and fbn2b were required for endocardial cell proliferation, endocardial Notch signaling and the proper formation of endocardial cell adhesion and tight junctions, all of which are crucial for cardiac valve development. We conclude that pku300 and fbn2b represent the few genes capable of regulating endocardial cell proliferation and signaling in zebrafish cardiac valve development.

Expression of the Lycat gene in the mouse cardiovascular and female reproductive systems.

The Lycat homologue in zebrafish maps to the deletion interval of the cloche mutant in which hematopoietic and endothelial cell lineages are affected. However, its definitive relationship to cloche is inconclusive, partly due to inadequate expression data of Lycat from any organisms. We precisely examined the temporal and spatial expression patterns of Lycat in mouse using RNA in situ hybridization, immunostaining, and BAC transgenesis. Lycat is initially expressed in developing heart, lung, and somites, and later becomes progressively restricted to all vascular smooth muscle cells. In adult ovaries, Lycat turns on in oocytes during the transition from primary to secondary follicles. Expression of the Lycat/reporter transgene in the extraembryonic mesoderm, cardiogenic mesoderm, and primitive streak, but not extraembryonic endoderm at E7.5, suggests its potential roles in regulating cardiac, smooth muscle, hematopoietic and endothelial lineages. Promoter mapping assay by transient transgenesis identifies a novel cardiac-specific regulatory region in the Lycat locus.

Alternative splicing of exon 10 in the tau gene as a target for treatment of tauopathies.

Tau aggregation is one of the major features in Alzheimer's disease and in several other tauopathies, including frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17), and progressive supranuclear palsy (PSP). More than 35 mutations in the tau gene have been identified from FTDP-17 patients. A group of these mutations alters splicing of exon 10, resulting in an increase in exon 10 inclusion into tau mRNA. Abnormal splicing with inclusion of exon 10 into tau mRNA has also been observed in PSP and AD patients. These results indicate that abnormal splicing of exon 10, leading to the production of tau with exon 10, is probably one of the mechanisms by which tau accumulates and aggregates in tauopathic brains. Therefore, modulation of exon 10 splicing in the tau gene could potentially be targeted to prevent tauopathies. To identify small molecules or compounds that could potentially be developed into drugs to treat tauopathies, we established a cell-based high-throughput screening assay. In this review, we will discuss how realistic, specific biological molecules can be found to regulate exon 10 splicing in the tau gene for potential treatment of tauopathies.

An acyltransferase controls the generation of hematopoietic and endothelial lineages in zebrafish.

Hematopoietic and endothelial cells develop from a common progenitor, the hemangioblast, or directly from mesodermal cells. The molecular pathway that regulates the specification of both cell lineages remains elusive. Here, we show that a lysocardiolipin acyltransferase, lycat, is critical for the establishment of both hematopoietic and endothelial lineages. We isolated lycat from the deletion interval of cloche, a zebrafish mutant that has dramatically reduced hematopoietic and endothelial cell lineages. Reduction of lycat mRNA levels in wild-type zebrafish embryos decreases both endothelial and hematopoietic lineages. Lycat mRNA rescues blood lineages in zebrafish cloche mutant embryos. E165R and G166L mutations in the highly conserved catalytic domain in lycat abolish its function in zebrafish hematopoiesis. Epistasis analysis supports that lycat acts upstream of scl and etsrp in zebrafish hemangioblast development. These data indicate that lycat is the earliest known player in the generation of both endothelial and hematopoietic lineages.

Emetine regulates the alternative splicing of Bcl-x through a protein phosphatase 1-dependent mechanism.

Exon 2 of the Bcl-x gene undergoes alternative splicing in which the Bcl-xS splice variant promotes apoptosis in contrast to the anti-apoptotic splice variant Bcl-xL. In this study, the regulation of the alternative splicing of pre-mRNA of Bcl-x was examined in response to emetine. Treatment of different types of cancer cells with emetine dihydrochloride downregulated the level of Bcl-xL mRNA with a concomitant increase in the mRNA level of Bcl-xS in a dose- and time-dependent manner. Pretreatment with calyculin A, an inhibitor of protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A), blocked emetine-induced alternative splicing in contrast to okadaic acid, a specific inhibitor of PP2A in cells, demonstrating a PP1-mediated mechanism. Our finding on the regulation of RNA splicing of members of the Bcl-2 family in response to emetine presents a potential target for cancer treatment.

BMPER is a conserved regulator of hematopoietic and vascular development in zebrafish.

For the proper development of vertebrate embryos as well as for survival of the adult organism, it is essential to form a functional vascular system. Molecules involved in this process are members of highly conserved families of proteins that exert conserved functions across species. Bone morphogenetic proteins (BMP) are extracellular factors that are regulated by extracellular modulators and bind to BMP receptors, which in turn activate intracellular signaling cascades. BMPs are necessary not only for induction of endothelial and hematopoietic lineages but also for further endothelial and hematopoietic cell differentiation. Previously, we identified BMPER (BMP endothelial cell precursor derived regulator) and demonstrated its spatiotemporal expression at sites of vasculogenesis and direct modulation of BMP activity. To directly investigate the role of BMPER in vascular development, we cloned the BMPER ortholog in zebrafish (zbmper). It is expressed at sites of high BMP activity, including vascular precursor cells located in the aortic arches and the intermediate cell mass during zebrafish embryonic development. Knockdown of zbmper results in a dorsalized phenotype, a reduced number of gata1 expressing hematopoietic precursor cells and of circulating blood cells as well as in a vascular phenotype. The generation of the caudal vein is compromised and the pattern guiding of the intersomitic vessels is disturbed, indicating that zbmper is required for early steps in vascular pattern formation and hematopoiesis in zebrafish.

Reprogramming of tau alternative splicing by spliceosome-mediated RNA trans-splicing: implications for tauopathies.

Frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) is caused by mutations in the gene encoding the microtubule-associated protein, tau. Some FTDP-17 mutations affect exon 10 splicing. To correct aberrant exon 10 splicing while retaining endogenous transcriptional control, we evaluated the feasibility of using spliceosome-mediated RNA trans-splicing (SMaRT) to reprogram tau mRNA. We designed a pre-trans-splicing molecule containing human tau exons 10 to 13 and a binding domain complementary to the 3' end of tau intron 9. A minigene comprising tau exons 9, 10, and 11 and minimal flanking intronic sequences was used as a target. RT-PCR analysis of SH-SY5Y cells or COS cells cotransfected with a minigene and a pre-trans-splicing molecule using primers to opposite sides of the predicted splice junction generated products containing exons 9 to 13. Sequencing of the chimeric products showed that an exact exon 9-exon 10 junction had been created, thus demonstrating that tau RNA can be reprogrammed by trans-splicing. Furthermore, by using the same paradigm with a minigene containing full-length intronic sequences, we show that cis-splicing exclusion of exon 10 can be by-passed by trans-splicing and that conversion of exon 10(-) tau RNA into exon 10(+) tau RNA could be achieved with approximately 34% efficiency. Our results demonstrate that an alternatively spliced exon can be replaced by trans-splicing and open the way to novel therapeutic applications of SMaRT for tauopathies and other disorders linked to aberrant alternative splicing.

RING finger ubiquitin-protein isopeptide ligase Nrdp1/FLRF regulates parkin stability and activity.

Parkin is a ubiquitin-protein isopeptide ligase. It has been suggested that loss of function in parkin causes accumulation and aggregation of its substrates, leading to death of dopaminergic neurons in Parkinson disease. Using the yeast two-hybrid screen, we isolated a RING finger protein that interacted with the N terminus of parkin in a Drosophila cDNA library. Interaction between human parkin and the mammalian RING finger protein homologue Nrdp1/FLRF, a ubiquitin-protein isopeptide ligase that ubiquitinates ErbB3 and ErbB4, was validated by in vitro binding assay, co-immunoprecipitation, and immunofluorescence co-localization. Significantly, pulse-chase experiments showed that cotransfection of Nrdp1 and parkin reduced the half-life of parkin from 5 to 2.5 h. Consistent with these findings, we further observed that degradation of CDCrel-1, a parkin substrate, was facilitated by overexpression of parkin protein. However, co-transfection of Nrdp1 with parkin reversed the effects of parkin on CDCrel-1 degradation. We conclude that Nrdp1 is a parkin modifier that accelerates degradation of parkin, resulting in a reduction of parkin activity.

A minimal length between tau exon 10 and 11 is required for correct splicing of exon 10.

Mutations that stimulate exon 10 inclusion into the human tau mRNA cause frontotemporal dementia with parkinsonism, associated with chromosome 17 (FTDP-17), and other tauopathies. This suggests that the ratio of exon 10 inclusion to exclusion in adult brain is one of the factors to determine biological functions of the tau protein. To investigate the underlying splicing mechanism and identify potential therapeutic targets for tauopathies, we generated a series of mini-gene constructs with intron deletions from the full length of tau exons 9-11 mini-gene construct. RT-PCR results demonstrate that there is a minimum distance requirement between exon 10 and 11 for correct splicing of the exon 10. In addition, SRp20, a member of serine-arginine (SR) protein family of splicing factors was found to facilitate exclusion of exon 10 in a dosage-dependent manner. Significantly, SRp20 also induced exon 10 skipping from pre-mRNAs containing mutations identified in FTDP-17 patients. Based on those results, we generated a cell-based system to measure inclusion to exclusion of exon 10 in the tau mRNA using the luciferase reporter. The firefly luciferase was fused into exon 11 in frame, and a stop code was also created in exon 10. Inclusion of exon 10 prevents luciferase expression, whereas exclusion of exon 10 generates luciferase activity. To minimize baseline luciferase expression, our reporter construct also contains a FTDP-17 mutation that increases exon 10 inclusion. We demonstrate that the splicing pattern of our reporter construct mimics that of endogenous tau gene. Co-transfection of SRp20 and SRp55, two SR proteins that promote exon 10 exclusion, increases production of luciferase. We conclude that this cell-based system can be used to identify biological substances that modulate exon 10 splicing.

Molecular Cloning of Human beta-arrestin1 cDNA, Expression and Functional Study.

Complete coding sequences of beta-arrestin1 (1A and 1B) were cloned through application of bioinformatics analysis to the dbEST database. beta-arrestin1A was overexpressed in E.coli with partial expression products as inclusion body. Anti-beta-arrestin1 antibodies were prepared by using purified inclusion body. Results also demonstrate that activation of inhibitory G protein mediated by delta and kappa pioid receptors was strongly attenuated by overexpression of beta-arrestin1A in co-transfected 293 cells.

Effects of Deleting the Amino-terminal Domain of GRK-2 on Its Function.

To reveal the possible role of the amino-terminal domain of G protein-coupled receptor kinases(GRKs)in receptor phosphorylation and/or modulation of its kinase activity, a truncated mutant of GRK-2 lacking the amino-terminal domain(deltaN-GRK2)was made. deltaN-GRK2 was expressed effectively in E.coli as a GST fusion protein and was purified by affinity chromatography on a GSH-Sepharose column. deltaN-GRK2 was then separated from GST tag by thrombin cleavage and recovered. Although deltaN-GRK2 had nearly identical activity with wild-type GRK-2 in phosphorylation of peptide substrate, it completely lost the ability to phosphorylate the light-activated receptor rhodopsin. Furthermore, deletion of the amino-terminal domain rendered GRK-2 unresponsive to the regulation of kinase activity by a truncated form of rhodopsin, (329)G-Rho(*) and beta gamma subunits of G protein. These results demonstrated that the amino-terminal domain was necessary to GRK2 for both the phosphorylation of receptor and the regulation of its kinase activity by the receptor. It was reasonable to postulate that this domain has little, if any effect on the catalytic domain of natural form of GRK2.