Biochemical identification of new proteins involved in splicing repression at the Drosophila P-element exonic splicing silencer

Splicing of the Drosophila P-element third intron (IVS3) is repressed in somatic tissues due to the function of an exonic splicing silencer (ESS) complex present on the 5' exon RNA. To comprehensively characterize the mechanisms of this alternative splicing regulation, we used biochemical fractionation and affinity purification to isolate the silencer complex assembled in vitro and identify the constituent proteins by mass spectrometry. Functional assays using splicing reporter minigenes identified the proteins hrp36 and hrp38 and the cytoplasmic poly(A)-binding protein PABPC1 as novel functional components of the splicing silencer. hrp48, PSI, and PABPC1 have high-affinity RNA-binding sites on the P-element IVS3 5' exon, whereas hrp36 and hrp38 proteins bind with low affinity to the P-element silencer RNA. RNA pull-down and immobilized protein assays showed that hrp48 protein binding to the silencer RNA can recruit hrp36 and hrp38. These studies identified additional components that function at the P-element ESS and indicated that proteins with low-affinity RNA-binding sites can be recruited in a functional manner through interactions with a protein bound to RNA at a high-affinity binding site. These studies have implications for the role of heterogeneous nuclear ribonucleoproteins (hnRNPs) in the control of alternative splicing at cis-acting regulatory sites

The microtubule-associated protein tau cross-links to two distinct sites on each α and β tubulin monomer via separate domains

The interaction between tubulin subunits and microtubule-associated proteins (MAPs) such as tau is fundamental for microtubule structure and function. Previous work has suggested that the 'microtubule biding domain' of tau (composed of three or four imperfect 18-amino acid repeats, separated by 13- or 14-amino acid inter-repeated regions) can bind to the C-terminal ends of both α and β tubulin monomers. Here, using covalent cross-linking strategies, we demonstrate that there are two distinct tau cross-linking sites (designated as 'C-terminal' and 'internal') on each α and β tubulin monomer. The C-terminal tau cross-linking site is located within the 12 C- terminal amino acids of both α and β tubulin, while the internal tau cross- linking site is located within the C-terminal one-third of α and β tubulin but not within the last 12 amino acids. In addition, we show that tau cross- links to the C-terminal site via its repeat 1 and/or the R1-R2 inter-repeat. The cross-linking of tau to the internal site is mediated by some subset of its other repeat units. Integrating these and earlier data with the 3.7 Å resolution model of the αβ tubulin dimer recently presented by E. Nogales et al. [(1998), Nature 391, 199-203], we propose a new model for the tau- microtubule interaction.Dr. J. M. Andreu (Centro de Investigaciones Biológicas, for supporting the generation of monoclonal antibodies P12E11, P5C3, and P6B11; Grant DGES PB95 0116

Prevention of Human Rhinovirus Infection by Multivalent Fab Molecules Directed against ICAM-1

We have developed a technology for improving avidity by making bivalent, trivalent, or tetravalent recombinant polypeptides. We designed tripartite proteins consisting of the Fab fragment of an antibody fused with a hinge derived from human immunoglobulin D that was further linked to polymerization domains derived from human coiled-coil proteins. We report here on the application of this method with a Fab domain directed against the major human rhinovirus receptor, intercellular adhesion molecule 1 (ICAM-1). Multivalent anti-ICAM-1 molecules were produced in bacteria and purified as soluble preassembled homogeneous proteins at high yield. These proteins successfully blocked rhinovirus infection in vitro, with the efficiency increasing from monomer to dimer, trimer, and tetramer. The diminished dissociation rate of these multivalent antibodies and their improved efficacy in preventing rhinovirus infection provide a foundation for producing prophylactic and therapeutic molecules against human rhinovirus, the causative agent of the majority of common colds

Aurora B-dependent Ndc80 degradation regulates kinetochore composition in meiosis.

The kinetochore complex is a conserved machinery that connects chromosomes to spindle microtubules. During meiosis, the kinetochore is restructured to accommodate a specialized chromosome segregation pattern. In budding yeast, meiotic kinetochore remodeling is mediated by the temporal changes in the abundance of a single subunit called Ndc80. We previously described the regulatory events that control the timely synthesis of Ndc80. Here, we report that Ndc80 turnover is also tightly regulated in meiosis: Ndc80 degradation is active in meiotic prophase, but not in metaphase I. Ndc80 degradation depends on the ubiquitin ligase APCAma1 and is mediated by the proteasome. Importantly, Aurora B-dependent Ndc80 phosphorylation, a mark that has been previously implicated in correcting erroneous microtubule-kinetochore attachments, is essential for Ndc80 degradation in a microtubule-independent manner. The N terminus of Ndc80, including a 27-residue sequence and Aurora B phosphorylation sites, is both necessary and sufficient for kinetochore protein degradation. Finally, defects in Ndc80 turnover predispose meiotic cells to chromosome mis-segregation. Our study elucidates the mechanism by which meiotic cells modulate their kinetochore composition through regulated Ndc80 degradation, and demonstrates that Aurora B-dependent regulation of kinetochores extends beyond altering microtubule attachments

The Drosophila Splicing Factor PSI Is Phosphorylated by Casein Kinase II and Tousled-Like Kinase

<div><p>Alternative splicing of pre-mRNA is a highly regulated process that allows cells to change their genetic informational output. These changes are mediated by protein factors that directly bind specific pre-mRNA sequences. Although much is known about how these splicing factors regulate pre-mRNA splicing events, comparatively little is known about the regulation of the splicing factors themselves. Here, we show that the <i>Drosophila</i> splicing factor P element Somatic Inhibitor (PSI) is phosphorylated at at least two different sites by at minimum two different kinases, casein kinase II (CK II) and tousled-like kinase (tlk). These phosphorylation events may be important for regulating protein-protein interactions involving PSI. Additionally, we show that PSI interacts with several proteins in <i>Drosophila</i> S2 tissue culture cells, the majority of which are splicing factors.</p> </div

Biochemcial fractionation and analysis of the PSI kinase.

<p>A) Purified recombinant PSI and PSI purified from Kc cells was treated with calf intestinal phosphatase (CIP) and then visualized by immunoblotting. B) MS2 spectra identifying phosphopeptides found in PSI. B and Y series ions and neutral loss of phosphate are indicated. Inset: sequence of the phosphopeptide and SEQUEST statistics. MS3 spectra and corresponding spectra of unmodified peptides are given in supplemental <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056401#pone-0056401-g001" target="_blank">Figure 1</a>.</p

Protein-protein interactions of PSI.

<p>A) GST pulldown assay using PSI mutant proteins. GST-PSI fusion proteins carrying the serine to alanine PSI mutations were phosphorylated using purified human casein kinase II and incubated with Kc nuclear extract. The resulting glutathione resin eluates were analyzed by silver staining and mass spectrometry. B) Silver stain of PSI and interacting proteins following anti-polyoma and anti-PSI immunoprecipitations. The asterisk indicates antibody heavy chain. C) Immunoblot analysis of (B) using anti-PSI antibody. D) Mass spectrometry analysis of (B). Proteins identified as interacting with PSI and the number of peptides observed for each protein are listed.</p

Biochemical purification of Drosophila casein kinase II.

<p>A) Purification strategy for endogenous casein kinase II. B) Protein composition of peak fraction of activity from the final heparin column visualized by SDS-PAGE and silver-staining. Species identified as casein kinase II alpha and beta are labeled. Bands labeled with an asterisk correspond to contaminating keratin. C) <i>In vitro</i> kinase assay of PSI mutant proteins. Serine to alanine PSI mutant proteins were phosphorylated <i>in vitro</i> using the peak fraction of activity from the final heparin column and using purified recombinant human casein kinase II (NEB P6010S). Assays were visualized using autoradiography, and, to ensure equal protein loading, coomassie staining.</p