In contrast PI3K Inhibitor Library in vitro to inc null mutations, neuronally restricted RNAi against insomniac is not associated with

a decrease in longevity ( Figure 6B) despite a strong reduction in sleep ( Figure 4A). Although neuronal insomniac depletion reduces sleep less severely than the inc1 or inc2 mutations (Figures 3C and 4A), and does not completely eliminate Insomniac protein expressed in the head (data not shown), these results nonetheless indicate that an acute reduction in sleep can be uncoupled from reduced longevity. The predominant insomniac transcript contains five exons and a large 3′UTR ( Figures 2A and S4), and encodes a 211 amino acid protein. We also detected a rare transcript variant in which the last intron is retained ( Figure S4C), yielding a predicted protein three residues shorter and with a different terminal residue ( Figure S4E). The mobility of endogenous Insomniac protein is slightly less than 25 kD in western blots ( Figure 2F), consistent with the 24.3 kD predicted molecular weight. The only characterized motif within Insomniac is an N-terminal Bric-à-brac, Tramtrack, and Broad/Pox virus and Zinc finger (BTB/POZ) domain, an ∼95 residue motif that mediates self-association as well as interaction with this website heterologous proteins (Stogios et al.,

2005). Three highly conserved orthologs of Insomniac, KCTD2, KCTD5, and KCTD17, are present in humans and other vertebrates (Figure 7A and data not shown). Sequence alignment of Insomniac and these orthologs reveals divergent N and C termini flanking the shared BTB domain and a C-terminal block of homology that corresponds to a globular domain of unknown function (Dementieva et al., 2009). Insomniac exhibits >60% identity and >75% similarity to each of its orthologs. BTB proteins are classified on the basis of their sequence and structure into several distinct subfamilies (Stogios et al., 2005), with Insomniac and its orthologs grouped into the potassium Astemizole channel tetramerization domain (KCTD) subfamily. Although the KCTD is named for its initial characterization as a sequence

promoting the assembly of voltage-gated potassium (Kv) channels (Li et al., 1992 and Shen et al., 1993), Insomniac and its orthologs are shorter in size and structurally distinct from Kv channels, as they lack characteristic transmembrane and ion transport domains. Including Insomniac, seven nonchannel KCTD proteins are present in Drosophila. Twenty-three evolutionarily related, nonchannel KCTD proteins are found in humans ( Figure 7B). Many BTB proteins function as adaptors for Cul3 ubiquitin ligase complexes (Xu et al., 2003, Pintard et al., 2003 and Geyer et al., 2003), including several well-characterized proteins of the non-channel KCTD subfamily. KCTD13/BACURD1 and TNFAIP1/BACURD2 (Chen et al.