Polypyrimidine tract binding protein (hnRNP I) is possibly a conserved modulator of miRNA-mediated gene regulation.

MiRNAs can regulate gene expression through versatile mechanisms that result in increased or decreased expression of the targeted mRNA and it could effect the expression of thousands of protein in a particular cell. An increasing body of evidence suggest that miRNAs action can be modulated by proteins that bind to the same 3'UTRs that are targeted by miRNAs, suggesting that other factors apart from miRNAs and their target sites determine miRNA-modulation of gene expression. We applied an affinity purification protocol using biotinylated let-7 miRNA inhibitor to isolate proteins that are involved in let-7 mediated gene regulation that resulted in an affinity purification of Polypyrimidine Tract Binding protein (PTB). Here we show that PTB interacts with miRNAs and human Argonaute 2 (hAgo2) through RNA as well as identified potential mammalian cellular targets that are co-regulated by PTB and hAgo2. In addition, using genetic approach, we have demonstrated that PTB genetically interacts with Caenorhabditis elegans let-7 indicating a conserved role for PTB in miRNA-mediated gene regulation.

A potential role for calcium / calmodulin-dependent protein kinase-related peptide in neuronal apoptosis: in vivo and in vitro evidence.

Previously, we have established that a product of the doublecortin-like kinase (DCLK) gene, DCLK-short, is cleaved by caspases during serum deprivation. Subsequently, the N-terminal cleavage product of DCLK-short facilitates apoptosis in the neuroblastoma cell line NG108. As this N-terminal cleavage product is highly homologous to calcium/calmodulin-dependent protein kinase-related peptide (CARP), another DCLK gene splice variant, we aimed to determine the possible apoptotic properties of CARP in vivo and in vitro. We report highly specific CARP expression in apoptotic granule cells in the rat dentate gyrus after adrenalectomy relative to healthy granule cells. CARP is significantly upregulated in the suprapyramidal blade of the dentate gyrus, with varying levels of upregulation, depending on the extent of adrenalectomy-induced apoptosis. Similar to the caspase-cleaved N-terminus of DCLK-short, CARP overexpression itself facilitated apoptosis in serum-deprived NG108 cells. Furthermore, CARP facilitated polymerization of tubulin in vitro and was capable of interacting with growth factor receptor-bound protein 2, an intracellular protein involved in vesicle trafficking. Together, our data demonstrate a facilitating role for CARP in the apoptotic process in granule cell populations sensitive to adrenalectomy, and suggest that this proapoptotic effect is mediated by increasing the stability of the microtubule cytoskeleton.

Characterisation of Fmrp in zebrafish: evolutionary dynamics of the fmr1 gene.

Fragile X syndrome is the most common inherited form of mental retardation. It is caused by the lack of the Fragile X Mental Retardation Protein (FMRP), which is encoded by the FMR1 gene. Although Fmr1 knockout mice display some characteristics also found in fragile X patients, it is a complex animal model to study brain abnormalities, especially during early embryonic development. Interestingly, the ortholog of the FMR1 gene has been identified not only in mouse, but also in zebrafish (Danio rerio). In this study, an amino acid sequence comparison of FMRP orthologs was performed to determine the similar regions of FMRP between several species, including human, mouse, frog, fruitfly and zebrafish. Further characterisation of Fmrp has been performed in both adults and embryos of zebrafish using immunohistochemistry and western blotting with specific antibodies raised against zebrafish Fmrp. We have demonstrated a strong Fmrp expression in neurons of the brain and only a very weak expression in the testis. In brain tissue, a different distribution of the isoforms of Fmrp, compared to human and mouse brain tissue, was shown using western blot analysis. Due to the high similarity between zebrafish Fmrp and human FMRP and their similar expression pattern, the zebrafish has great potential as a complementary animal model to study the pathogenesis of the fragile X syndrome, especially during embryonic development.

Characterization of Fxr1 in Danio rerio; a simple vertebrate model to study costamere development.

The X-linked FMR1 gene, which is involved in the fragile X syndrome, forms a small gene family with its two autosomal homologs, FXR1 and FXR2. Mouse models for the FXR genes have been generated and proved to be valuable in elucidating the function of these genes, particularly in adult mice. Unfortunately, Fxr1 knockout mice die shortly after birth, necessitating an animal model that allows the study of the role of Fxr1p, the gene product of Fxr1, in early embryonic development. For gene function studies during early embryonic development the use of zebrafish as a model organism is highly advantageous. In this paper the suitability of the zebrafish as a model organism to study Fxr1p function during early development is explored. As a first step, we present here the initial characterization of Fxr1p in zebrafish. Fxr1p is present in all the cells from zebrafish embryos from the 2/4-cell stage onward; however, during late development a more tissue-specific distribution is found, with the highest expression in developing muscle. In adult zebrafish, Fxr1p is localized at the myoseptum and in costamere-like granules in skeletal muscle. In the testis, Fxr1p is localized in immature spermatogenic cells and in brain tissue Fxr1p displays a predominantly nuclear staining in neurons throughout the brain. Finally, the different tissue-specific isoforms of Fxr1p are characterized. Since the functional domains and the expression pattern of Fxr1p in zebrafish are comparable to those in higher vertebrates such as mouse and human, we conclude that the zebrafish is a highly suitable model for functional studies of Fxr1p.

Functional differences between two DCLK splice variants.

Recently, we have cloned two splice variants of the doublecortin-like kinase (DCLK) gene, called DCLK-short-A and -B, both of which encode calcium/calmodulin-dependent protein kinase (CaMK)-like proteins with different C-terminal ends. Using in situ hybridization, we have found that both are highly expressed in limbic structures of the brain and that their expression differs in a number of brain areas. DCLK-short-A is relatively more strongly expressed than DCLK-short-B in the subependymal zone. The DCLK-short-B variant shows stronger expression in the cortex, the ventromedial and dorsomedial hypothalamic nuclei, the arcuate nucleus, the zona incerta and the subincertal nucleus. Also, within the hippocampus, the relative distribution of these two splice variants differs. DCLK-short-B expression compared to DCLK-short-A is highest in the CA1 area. The expression of the A variant is highest in the CA3/CA4 area. Additionally, DCLK-short-B is expressed at a higher level than DCLK-short-A in the substantia nigra and the mammillary nucleus. Both DCLK-short-A and -B were located in the cytoplasm, however DCLK-short-B was also found specifically in growth cone like structures and near the nucleus. Both DCLK-short proteins phosphorylate autocamtide and syntide, two highly specific CaMK substrates. Finally, removal of the C-terminal end of DCLK-short leads to a 10-fold increase of kinase activity, indicating that the different C-termini represent auto-inhibitory domains. Our results indicate that DCLK-short-A and -B control different neuronal processes that overlap with those controlled by CaMKs.