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Five years after the completion of the sequence of the Drosophila melanogaster genome, the number of protein-coding genes it contains remains a matter of debate; the number of computational gene predictions greatly exceeds the number of validated gene annotations. We have assembled a collection of >10,000 gene predictions that do not overlap existing gene annotations and have developed a process for their validation that allows us to efficiently prioritize and experimentally validate predictions from various sources by sequencing RT-PCR products to confirm gene structures. Our data provide experimental evidence for 122 protein-coding genes. Our analyses suggest that the entire collection of predictions contains only ≈700 additional protein-coding genes. Although we cannot rule out the discovery of genes with unusual features that make them refractory to existing methods, our results suggest that the D. melanogaster genome contains ≈14,000 protein-coding genes.

microRNAs (miRNAs) are 21–22-nucleotide noncoding RNAs that are widely believed to regulate complementary mRNA targets. However, due to the modest amount of pairing involved, only a few out of the hundreds of known animal miRNAs have thus far been connected to mRNA targets. Here, we considered the possibility that miRNAs might regulate non-mRNA targets, namely other miRNAs. To do so, we conducted a systematic assessment of the nearly complete catalogs of animal miRNAs for potential miRNA:miRNA complements. Our analysis uncovered several compelling examples that strongly suggest a function for miRNA duplexes, thus adding a potential layer of regulatory sophistication to the small RNA world. Interestingly, the most striking examples involve miRNAs complementary to members of the K-box family and Brd-box family, two classes of miRNAs previously implicated in regulation of Notch target genes. We emphasize that patterns of nucleotide constraint indicate that miRNA complementarity is not a simple consequence of miRNA:miRNA* complementarity; however, our findings do suggest that the potential regulatory consequences of the latter also deserve investigation.