NF45 dimerizes with NF90, Zfr and SPNR via a conserved domain that has a nucleotidyltransferase fold

Nuclear factors NF90 and NF45 form a complex involved in a variety of cellular processes and are thought to affect gene expression both at the transcriptional and translational level. In addition, this complex affects the replication of several viruses through direct interactions with viral RNA. NF90 and NF45 dimerize through their common ‘DZF’ domain (domain associated with zinc fingers). NF90 has additional double-stranded RNA-binding domains that likely mediate its association with target RNAs. We present the crystal structure of the NF90/NF45 dimerization complex at 1.9-Å resolution. The DZF domain shows structural similarity to the template-free nucleotidyltransferase family of RNA modifying enzymes. However, both NF90 and NF45 have lost critical catalytic residues during evolution and are therefore not functional enzymes. Residues on NF90 that make up its interface with NF45 are conserved in two related proteins, spermatid perinuclear RNA-binding protein (SPNR) and zinc-finger RNA-binding protein (Zfr). Using a co-immunoprecipitation assay and site-specific mutants, we demonstrate that NF45 is also able to recognize SPNR and Zfr through the same binding interface, revealing that NF45 is able to form a variety of cellular complexes with other DZF-domain proteins

Assessing the Utility of Thermodynamic Features for microRNA Target Prediction under Relaxed Seed and No Conservation Requirements

BACKGROUND: Many computational microRNA target prediction tools are focused on several key features, including complementarity to 5'seed of miRNAs and evolutionary conservation. While these features allow for successful target identification, not all miRNA target sites are conserved and adhere to canonical seed complementarity. Several studies have propagated the use of energy features of mRNA:miRNA duplexes as an alternative feature. However, different independent evaluations reported conflicting results on the reliability of energy-based predictions. Here, we reassess the usefulness of energy features for mammalian target prediction, aiming to relax or eliminate the need for perfect seed matches and conservation requirement. METHODOLOGY/PRINCIPAL FINDINGS: We detect significant differences of energy features at experimentally supported human miRNA target sites and at genome-wide sites of AGO protein interaction. This trend is confirmed on datasets that assay the effect of miRNAs on mRNA and protein expression changes, and a simple linear regression model leads to significant correlation of predicted versus observed expression change. Compared to 6-mer seed matches as baseline, application of our energy-based model leads to ∼3-5-fold enrichment on highly down-regulated targets, and allows for prediction of strictly imperfect targets with enrichment above baseline. CONCLUSIONS/SIGNIFICANCE: In conclusion, our results indicate significant promise for energy-based miRNA target prediction that includes a broader range of targets without having to use conservation or impose stringent seed match rules

Tissue Type-Specific Expression of the dsRNA-Binding Protein 76 and Genome-Wide Elucidation of Its Target mRNAs

Background: RNA-binding proteins accompany all steps in the life of mRNAs and provide dynamic gene regulatory functions for rapid adjustment to changing extra-or intracellular conditions. The association of RNA-binding proteins with their targets is regulated through changing subcellular distribution, post-translational modification or association with other proteins. Methodology: We demonstrate that the dsRNA binding protein 76 (DRBP76), synonymous with nuclear factor 90, displays inherently distinct tissue type-specific subcellular distribution in the normal human central nervous system and in malignant brain tumors of glial origin. Altered subcellular localization and isoform distribution in malignant glioma indicate that tumor-specific changes in DRBP76-related gene products and their regulatory functions may contribute to the formation and/or maintenance of these tumors. To identify endogenous mRNA targets of DRBP76, we performed RNA-immunoprecipitation and genome-wide microarray analyses in HEK293 cells, and identified specific classes of transcripts encoding critical functions in cellular metabolism. Significance: Our data suggest that physiologic DRBP76 expression, isoform distribution and subcellular localization are profoundly altered upon malignant transformation. Thus, the functional role of DRBP76 in co- or post-transcriptional gene regulation may contribute to the neoplastic phenotype

The Legacies and Potentials of Feminism in Art de Appel Arts Centre, Amsterdam: A Case Study

This report is the outcome of a month-long practicum and exploratory study of the history of feminist art at de Appel arts centre, an internationally oriented arts center located in Amsterdam. The result of this study is a documentary film exploring the connections between Feministische Kunst Internationaal (Feminist Art International), a show held at de Appel in the winter of 1978-\u2779, and If I Can\u27t Dance, I Don\u27t Want To Be Part Of Your Revolution (IICD) Edition III “Masquerade,” a rolling curatorial platform working in collaboration with de Appel in the fall of 2008. Data was obtained by means of qualitative methods including participant observation, direct observation and focused interviews, and through intensive historical research utilizing the vast array of materials in de Appel\u27s archive. De Appel arts centre has been, and continues to be, a center focused on exposing the public to the latest and most contemporary developments in the art world. The choice to host Feministische Kunst Internationaal in 1978 worked to validate feminist art and the contributions of women-artists at a time when the field was overwhelmingly patriarchal, sexist, and inaccessible to women. Thirty years later, “If I Can\u27t Dance...”, in collaboration with de Appel, chose to once again revisit and explore the notions of performativity, agency, empowerment, enactment, and gesture that stem from the legacies of the feminist movement. By refusing to focus on static art forms and by incorporating a vast variety of mediums, publications, and discursive events in their exhibitions, de Appel continues to work to facilitate and encourage much-needed further discussion, thinking, and debate about the topics they explore. It is concluded that the themes and issues feminist artists dealt with in the late 1970s, as well as the surrounding theoretical debates about the nature and role of feminism, are all still extremely relevant today. I conclude that it is vital to re-examine the history of feminism, and especially the history of feminist art because of the vast number of relevant ideas and debates that are still unresolved. Perhaps by looking back and examining our history, we can once again begin a discussion about the relevance of feminism and feminist ideas today. Suggestions for future research include the effectiveness of art as a political tool in the realm of feminism/gender, art as social activism, the degree of public accessibility and knowledge to new developments in the art world, or a study of public reactions to the variety of mediums employed in future editions of “If I Can\u27t Dance...”

BRUGADA SYNDROME AND CONDUCTION SYSTEM DISEASE ARE LINKED TO A SINGLE SODIUM CHANNEL MUTATION.

The function of the 12 positive charges in the 53-residue III/IV interdomain linker of the cardiac Na(+) channel is unclear. We have identified a four-generation family, including 17 gene carriers with long QT syndrome, Brugada syndrome, and conduction system disease with deletion of lysine 1500 (DeltaK1500) within the linker. Three family members died suddenly. We have examined the functional consequences of this mutation by measuring whole-cell and single-channel currents in 293-EBNA cells expressing the wild-type and DeltaK1500 mutant channel. The mutation shifted V(1/2)h( infinity ) to more negative membrane potentials and increased k(h) consistent with a reduction of inactivation valence of 1. The shift in h( infinity ) was the result of an increase in closed-state inactivation rate (11-fold at -100 mV). V(1/2)m was shifted to more positive potentials, and k(m) was doubled in the DeltaK1500 mutant. To determine whether the positive charge deletion was the basis for the gating changes, we performed the mutations K1500Q and K1500E (change in charge, -1 and -2, respectively). For both mutations, V(1/2)h was shifted back toward control; however, V(1/2)m shifted progressively to more positive potentials. The late component of Na(+) current was increased in the DeltaK1500 mutant channel. These changes can account for the complex phenotype in this kindred and point to an important role of the III/IV linker in channel activation

Long QT syndrome, Brugada syndrome, and conduction system disease are linked to a single sodium channel mutation

The function of the 12 positive charges in the 53-residue III/IV interdomain linker of the cardiac Na(+) channel is unclear. We have identified a four-generation family, including 17 gene carriers with long QT syndrome, Brugada syndrome, and conduction system disease with deletion of lysine 1500 (ΔK1500) within the linker. Three family members died suddenly. We have examined the functional consequences of this mutation by measuring whole-cell and single-channel currents in 293-EBNA cells expressing the wild-type and ΔK1500 mutant channel. The mutation shifted V(1/2)h(∞) to more negative membrane potentials and increased k(h) consistent with a reduction of inactivation valence of 1. The shift in h(∞) was the result of an increase in closed-state inactivation rate (11-fold at –100 mV). V(1/2)m was shifted to more positive potentials, and k(m) was doubled in the ΔK1500 mutant. To determine whether the positive charge deletion was the basis for the gating changes, we performed the mutations K1500Q and K1500E (change in charge, –1 and –2, respectively). For both mutations, V(1/2)h was shifted back toward control; however, V(1/2)m shifted progressively to more positive potentials. The late component of Na(+) current was increased in the ΔK1500 mutant channel. These changes can account for the complex phenotype in this kindred and point to an important role of the III/IV linker in channel activation

Improved adenoviral vector for vascular gene therapy : beneficial effects on vascular function and inflammation.

First-generation, E1-deleted adenoviral vectors (E1-AV) can transduce the vascular endothelium with high efficiency, but their use is limited by the resulting acute endothelial injury and the long-term development of intimal hyperplasia. To reduce the impact of viral proteins on the gene-modified cells, a second-generation adenoviral vector with an additional pair of deletions in the E4 region was developed. To determine whether this E1/E4-AV vector would be useful for vascular gene transfer, we directly compared the efficiency of gene transfer to uninjured rabbit carotid arteries using either an E1/E4-AV or an E1-AV vector encoding beta-galactosidase. Both vectors efficiently transduced vascular endothelium; however, the E1/E4-AV vector gene-modified vessels showed higher beta-galactosidase expression 10 days after gene transfer. Importantly, the E1/E4-AV vector produced substantially less endothelial cell activation, less inflammation, and reduced neointimal hyperplasia compared with the E1-AV vector-treated vessels. The E1-AV vector-transduced vessels also demonstrated significantly impaired endothelium-dependent relaxation whereas the E1/E4-AV vector did not impact vasomotor function, even at doses of virus in 5-fold excess of the amount required for >90% transduction of the endothelium. We conclude that the E1/E4-AV vector is superior to the E1-AV vector for vascular gene therapy because of the prolonged transgene expression, reduced vascular inflammation, reduced intimal hyperplasia, and maintenance of normal vasomotor function