Proteomic diversity is frequently achieved by alternative RNAsplicing events that can be fine-tuned in tissue-specific and developmentally regulated ways. Understanding this type of genetic regulation is compelling because of the extensive complexity of alternative splicing found in the nervous system. quaking (qk), one of the classical mouse dysmyelination mutants, is defective for the expression of myelin-associated glycoprotein (MAG), and the misregulation of MAG pre-mRNA alternative splicing is implicated as a causal factor. The qk locus encodes several RNA-binding proteins with heterogeneous nuclear ribonucleoprotein K-type homology, a characteristic of several known alternative splicing regulators. Here we test the nuclear-localized qk isoform (QKI-5) for its ability to regulate alternative splicing of MAG pre-mRNA in transient coexpression assays. QKI-5 exhibits properties of a negative regulator of MAG exon 12 alternative splicing. An intronic sequence element required for the repressive function and binding of QKI-5 is also identified. Direct evidence for irregularities in alternative splicing of MAG and other myelin protein transcripts in the qk mouse is demonstrated.A lternative splicing is a powerful way to regulate gene expression at the posttranscriptional level and to generate protein diversity (1, 2). Understanding the molecular basis of such mechanisms is an urgent challenge, because the number of protein-coding genes in humans has been estimated at barely double the number in Drosophila and Caenorhabditis elegans. Between 35% and 59% of human genes are predicted to generate alternatively spliced mRNA isoforms (3).Mouse quaking (qk), also known as quaking viable (qk v ), is a classical recessive dysmyelination mutation (4). The qk locus produces a diverse set of proteins by alternative splicing (5, 6). They all contain a single heterogeneous nuclear ribonucleoprotein K homology (KH) RNA-binding domain and belong to the evolutionarily conserved signal transduction and activator of RNA (STAR) family (7,8). The first three studied in detail are constructed with the same 311-aa body, but have different carboxyl tails. QKI-5 is the only nuclear isoform and shuttles between the nucleus and cytoplasm (9, 10). The expression of QKI isoforms is developmentally regulated, with QKI-5 being highly expressed throughout the embryogenesis and neonatal stages and decreasing gradually thereafter (7, 9). In postnatal day 14 (P14) mutant mice QKI proteins are decreased exclusively in myelin-forming cells. In addition, the QKI-5 expression level in qk v brain correlates with the severity of dysmyelinating phenotype, suggesting a function of QKI-5 in myelination (9).The relationship between the decreased QKI protein in affected mice and their myelination defects is not understood. It has been shown that several myelin-specific genes are alternatively spliced, including myelin basic protein (MBP), proteolipid protein (PLP), and MAG (11-13). Some splicing events seem to be abnormal in qk v mice. The best-documented c...

Patients with clinical manifestations of leishmaniasis, including cutaneous leishmaniasis, have limited treatment options, and existing therapies frequently have significant untoward liabilities. Rapid expansion in the diversity of available cutaneous leishmanicidal chemotypes is the initial step in finding alternative efficacious treatments. To this end, we combined a low-stringency Leishmania major promastigote growth inhibition assay with a structural computational filtering algorithm. After a rigorous assay validation process, we interrogated ∼200,000 unique compounds for L. major promastigote growth inhibition. Using iterative computational filtering of the compounds exhibiting >50% inhibition, we identified 553 structural clusters and 640 compound singletons. Secondary confirmation assays yielded 93 compounds with EC50s ≤ 1 µM, with none of the identified chemotypes being structurally similar to known leishmanicidals and most having favorable in silico predicted bioavailability characteristics. The leishmanicidal activity of a representative subset of 15 chemotypes was confirmed in two independent assay formats, and L. major parasite specificity was demonstrated by assaying against a panel of human cell lines. Thirteen chemotypes inhibited the growth of a L. major axenic amastigote-like population. Murine in vivo efficacy studies using one of the new chemotypes document inhibition of footpad lesion development. These results authenticate that low stringency, large-scale compound screening combined with computational structure filtering can rapidly expand the chemotypes targeting in vitro and in vivo Leishmania growth and viability.

The polypyrimidine tract binding protein (PTB, or hnRNP I) contains four RNA-binding domains of the ribonucleoprotein fold type (RRMs 1, 2, 3, and 4), and mediates the negative regulation of alternative splicing through sequencespecific binding to intronic splicing repressor elements. To assess the roles of individual RRM domains in splicing repression, a neural-specific splicing extract was used to screen for loss-of-function mutations that fail to switch splicing from the neural to nonneural pathway. These results show that three RRMs are sufficient for wild-type RNA binding and splicing repression activity, provided that RRM4 is intact. Surprisingly, the deletion of RRM4, or as few as 12 RRM4 residues, effectively uncouples these functions. Such an uncoupling phenotype is unique to RRM4, and suggests a possible regulatory role for this domain either in mediating specific RNA contacts, and/or contacts with putative splicing corepressors. Evidence of a role for RRM4 in anchoring PTB binding adjacent to the branch site is shown by mobility shift and RNA footprinting assays.