ELAV is a neuron-specific RNA-binding protein in Drosophila that is required for development and maintenance of neurons. ELAV regulates alternative splicing of Neuroglian and erect wing (ewg) transcripts, and has been shown to form a multimeric complex on the last ewg intron. The protein has three RNA recognition motifs (RRM1, 2 and 3) with a hinge region between RRM2 and 3. In this study, we used the yeast two-hybrid system to determine the multimerization domain of ELAV. Using deletion constructs, we mapped an interaction activity to a region containing most of RRM3. We found three conserved short sequences in RRM3 that were essential for the interaction, and also sufficient to give the interaction activity to RRM2 when introduced into it. In our in vivo functional assay, a mutation in one of the three sequences showed reduced activity in splicing regulation, underlining the functional importance of multimerization. However, RRM2 with the three RRM3 interaction sequences did not function as RRM3 in vivo, which suggested that multimerization is not the only function of RRM3. Our results are consistent with a model in which RRM3 serves as a bi-functional domain that interacts with both RNA and protein.

Sexual recognition among individuals is crucial for the reproduction of animals. In Drosophila, like in many other animals, pheromones are suggested to play an important role in conveying information about an individual, such as sex, maturity and mating status. Sex-specific cuticular hydrocarbon components are thought to be major sex pheromones in Drosophila, and are postulated to act through the gustatory system, since they are mostly non-volatile chemicals. However, very little is known about the molecular and neural bases of gustatory pheromone reception. So far, a few putative gustatory receptors, including Gr32a and Gr68a, have been implicated in courtship behavior. Here, we examine another putative gustatory receptor, Gr39a, which shares a cluster with both Gr32a and Gr68a in a molecular phylogeny of the gustatory receptor family, for its potential role in courtship behavior. The Gr39a gene produces four isoforms through alternative splicing of different 5'-most exons. A quantitative real-time PCR analysis showed that the expression levels of all four splice variants of Gr39a were reduced in a fly line in which a P element was inserted into the Gr39a locus. Homozygous and hemizygous males for the P-element insertion, as well as males in which Gr39a was knocked down by RNAi, all showed reduced courtship levels toward females. The courtship levels returned to normal when the P element was excised out. A close analysis of courtship behavior of the mutant males revealed that the average duration of a continuous courtship bout was significantly shorter in the mutants than in the wild type. The results suggest that Gr39a has a role in sustaining courtship behavior in males, possibly through the reception of a stimulating arrestant pheromone.

No abstract

In Drosophila, some neurons develop sex-specific neurites that contribute to dimorphic circuits for sex-specific behavior. As opposed to the idea that the sexual dichotomy in transcriptional profiles produced by a sex-specific factor underlies such sex differences, we discovered that the sex-specific cleavage confers the activity as a sexual-fate inducer on the pleiotropic transcription factor Longitudinals lacking (Lola). Surprisingly, Fruitless, another transcription factor with a master regulator role for courtship circuitry formation, directly binds to Lola to protect its cleavage in males. We also show that Lola cleavage involves E3 ubiquitin ligase Cullin1 and 26S proteasome. Our work adds a new dimension to the study of sex-specific behavior and its circuit basis by unveiling a mechanistic link between proteolysis and the sexually dimorphic patterning of circuits. Our findings may also provide new insights into potential causes of the sex-biased incidence of some neuropsychiatric diseases and inspire novel therapeutic approaches to such disorders.

We have constructed a P-element-based gene search vector for efficient detection of genes in Drosophila melanogaster. The vector contains two copies of the upstream activating sequence (UAS) enhancer adjacent to a core promoter, one copy near the terminal inverted repeats at each end of the vector, and oriented to direct transcription outward. Genes were detected on the basis of phenotypic changes caused by GAL4-dependent forced expression of vector-flanking DNA, and the transcripts were identified with reverse transcriptase PCR (RT-PCR) using the vector-specific primer and followed by direct sequencing. The system had a greater sensitivity than those already in use for gain-of-function screening: 64% of the vector insertion lines (394/613) showed phenotypes with forced expression of vector-flanking DNA, such as lethality or defects in adult structure. Molecular analysis of 170 randomly selected insertions with forced expression phenotypes revealed that 21% matched the sequences of cloned genes, and 18% matched reported expressed sequence tags (ESTs). Of the insertions in cloned genes, 83% were upstream of the protein-coding region. We discovered two new genes that showed sequence similarity to human genes, Ras-related protein 2 and microsomal glutathione S-transferase. The system can be useful as a tool for the functional mapping of the Drosophila genome.