Alternative splicing is thought to be regulated by nonspliceosomal RNA binding proteins that modulate the association of core components of the spliceosome with the pre-mRNA. Although the majority of metazoan genes encode pre-mRNAs that are alternatively spliced, remarkably few splicing regulators are currently known. Here, we used RNA interference to examine the role of >70% of the Drosophila RNA-binding proteins in regulating alternative splicing. We identified 47 proteins as splicing regulators, 26 of which have not previously been implicated in alternative splicing. Many of the regulators we identified are nonspliceosomal RNA-binding proteins. However, our screen unexpectedly revealed that altering the concentration of certain core components of the spliceosome specifically modulates alternative splicing. These results significantly expand the number of known splicing regulators and reveal an extraordinary richness in the mechanisms that regulate alternative splicing. P re-mRNA splicing involves the removal of introns and ligation of the flanking exons. This reaction is catalyzed by the spliceosome, a macromolecular machine composed of five RNAs and hundreds of proteins (1). Alternative splicing generates multiple mRNAs from a single gene, thus increasing proteome diversity (2). Alternative splicing also plays a key role in the regulation of gene expression in many developmental processes ranging from sex determination to apoptosis (3), and defects in alternative splicing have been linked to many human disorders (4). In general, alternative splicing is regulated by proteins that associate with the pre-mRNA and function to either enhance or repress the ability of the spliceosome to recognize the splice site(s) flanking the regulated exon (5). Whether a particular alternative exon will be included or excluded from an mRNA in each cell is thought to be determined by the relative concentration of a number of positive and negative splicing regulators and the interactions of these factors with the pre-mRNA and components of the spliceosome (5).Although at least 74% of human genes encode alternatively spliced mRNAs (6), relatively few splicing regulators have been identified. Much of our insight into the mechanisms of splicing regulation was initially obtained by genetic analysis of the sex determination pathway in Drosophila (3). These experiments have identified three proteins, Sex-lethal (SXL), Transformer (TRA), and Transformer 2 (TRA2), that tightly regulate the alternative splicing of five genes, Sex-lethal, transformer, male specific lethal-2, fruitless, and doublesex. Subsequent biochemical experiments helped to elucidate the mechanisms by which SXL, TRA, and TRA2 function in this pathway. Aside from these examples, a simple genetic system to analyze specific alternative splicing events has not been available. Here we describe an RNA interference (RNAi) screen in cultured Drosophila cells designed to identify RNA-binding proteins that regulate alternative splicing of pre-mRNAs transcribed from endogenous ge...

The plasma membrane targeting of Lgl, a key polarity and tumor suppressor protein, is mediated by electrostatic interactions between a polybasic motif in Lgl and phospholipids on the plasma membrane, and this mechanism is regulated by hypoxia and aPKC-phosphorylation.

Heritable mutations, known as inborn errors of metabolism, cause numerous devastating human diseases, typically as a result of a deficiency in essential metabolic products or the accumulation of toxic intermediates. We have isolated a missense mutation in the Drosophila sugarkill (sgk) gene that causes phenotypes analogous to symptoms of triosephosphate isomerase (TPI) deficiency, a human familial disease, characterized by anaerobic metabolic dysfunction resulting from pathological missense mutations affecting the encoded TPI protein. In Drosophila, the sgk gene encodes the glycolytic enzyme TPI. Our analysis of sgk mutants revealed TPI impairment associated with reduced longevity, progressive locomotor deficiency, and neural degeneration. Biochemical studies demonstrate that mutation of this glycolytic enzyme gene does not result in a bioenergetic deficit, suggesting an alternate cause of enzymopathy associated with TPI impairment.

Mitochondrial encephalomyopathies are common and devastating multisystem genetic disorders characterized by neuromuscular dysfunction and tissue degeneration. Point mutations in the human mitochondrial ATP6 gene are known to cause several related mitochondrial disorders: NARP (neuropathy, ataxia, and retinitis pigmentosa), MILS (maternally inherited Leigh's syndrome), and FBSN (familial bilateral striatal necrosis). We identified a pathogenic mutation in the Drosophila mitochondrial ATP6 gene that causes progressive, adult-onset neuromuscular dysfunction and myodegeneration. Our results demonstrate ultrastructural defects in the mitochondrial innermembrane, neural dysfunction, and a marked reduction in mitochondrial ATP synthase activity associated with this mutation. This Drosophila mutant recapitulates key features of the human neuromuscular disorders enabling detailed in vivo studies of these enigmatic diseases.

SummaryTriosephosphate isomerase (TPI) is a glycolytic enzyme that converts dihydroxyacetone phosphate (DHAP) into glyceraldehyde 3-phosphate (GAP). Glycolytic enzyme dysfunction leads to metabolic diseases collectively known as glycolytic enzymopathies. Of these enzymopathies, TPI deficiency is unique in the severity of neurological symptoms. The Drosophila sugarkill mutant closely models TPI deficiency and encodes a protein prematurely degraded by the proteasome. This led us to question whether enzyme catalytic activity was crucial to the pathogenesis of TPI sugarkill neurological phenotypes. To study TPI deficiency in vivo we developed a genomic engineering system for the TPI locus that enables the efficient generation of novel TPI genetic variants. Using this system we demonstrate that TPI sugarkill can be genetically complemented by TPI encoding a catalytically inactive enzyme. Furthermore, our results demonstrate a non-metabolic function for TPI, the loss of which contributes significantly to the neurological dysfunction in this animal model.