Plasmid-based gap-repair recombineered transgenes reveal a central role for introns in mutually exclusive alternative splicing in Down Syndrome Cell Adhesion Molecule exon 4.

Alternative splicing is a key feature of human genes, yet studying its regulation is often complicated by large introns. The Down Syndrome Cell Adhesion Molecule (Dscam) gene from Drosophila is one of the most complex genes generating vast molecular diversity by mutually exclusive alternative splicing. To resolve how alternative splicing in Dscam is regulated, we first developed plasmid-based UAS reporter genes for the Dscam variable exon 4 cluster and show that its alternative splicing is recapitulated by GAL4-mediated expression in neurons. We then developed gap-repair recombineering to very efficiently manipulate these large reporter plasmids in Escherichia coli using restriction enzymes or sgRNA/Cas9 DNA scission to capitalize on the many benefits of plasmids in phiC31 integrase-mediated transgenesis. Using these novel tools, we show that inclusion of Dscam exon 4 variables differs little in development and individual flies, and is robustly determined by sequences harbored in variable exons. We further show that introns drive selection of both proximal and distal variable exons. Since exon 4 cluster introns lack conserved sequences that could mediate robust long-range base-pairing to bring exons into proximity for splicing, our data argue for a central role of introns in mutually exclusive alternative splicing of Dscam exon 4 cluster.

Concentration and Localization of Coexpressed ELAV/Hu Proteins Control Specificity of mRNA Processing.

Neuronally coexpressed ELAV/Hu proteins comprise a family of highly related RNA binding proteins which bind to very similar cognate sequences. How this redundancy is linked to in vivo function and how gene-specific regulation is achieved have not been clear. Analysis of mutants in Drosophila ELAV/Hu family proteins ELAV, FNE, and RBP9 and of genetic interactions among them indicates that they have mostly independent roles in neuronal development and function but have converging roles in the regulation of synaptic plasticity. Conversely, ELAV, FNE, RBP9, and human HuR bind ELAV target RNA in vitro with similar affinities. Likewise, all can regulate alternative splicing of ELAV target genes in nonneuronal wing disc cells and substitute for ELAV in eye development upon artificially increased expression; they can also substantially restore ELAV's biological functions when expressed under the control of the elav gene. Furthermore, ELAV-related Sex-lethal can regulate ELAV targets, and ELAV/Hu proteins can interfere with sexual differentiation. An ancient relationship to Sex-lethal is revealed by gonadal expression of RBP9, providing a maternal fail-safe for dosage compensation. Our results indicate that highly related ELAV/Hu RNA binding proteins select targets for mRNA processing through alteration of their expression levels and subcellular localization but only minimally by altered RNA binding specificity.

Regulation of ELAV/Hu RNA-binding proteins by phosphorylation.

ELAV (embryonic lethal/abnormal visual system)/Hu proteins comprise a family of highly related neuronal RBPs (RNA-binding proteins) involved in many aspects of mRNA processing. Although they bind to highly similar short sequence motifs, they have acquired diverse functions suggesting that cellular signalling is important for their functional diversification. Indeed, ELAV/Hu proteins harbour many phosphorylatable amino acids. In the present article, we review our current knowledge about phosphorylation of ELAV/Hu proteins and how phosphorylation affects cellular localization of ELAV/Hu proteins and their binding to RNA.

Correlating structure and ligand affinity in drug discovery: a cautionary tale involving second shell residues.

A high-resolution crystallographic structure determination of a protein-ligand complex is generally accepted as the 'gold standard' for structure-based drug design, yet the relationship between structure and affinity is neither obvious nor straightforward. Here we analyze the interactions of a series of serine proteinase inhibitors with trypsin variants onto which the ligand-binding site of factor Xa has been grafted. Despite conservative mutations of only two residues not immediately in contact with ligands (second shell residues), significant differences in the affinity profiles of the variants are observed. Structural analyses demonstrate that these are due to multiple effects, including differences in the structure of the binding site, differences in target flexibility and differences in inhibitor binding modes. The data presented here highlight the myriad competing microscopic processes that contribute to protein-ligand interactions and emphasize the difficulties in predicting affinity from structure.