Revisiting the protein-coding gene catalog ofDrosophila melanogasterusing 12 fly genomes

Abstract

The availability of sequenced genomes from 12 Drosophila species has enabled the use of comparative genomics for the systematic discovery of functional elements conserved within this genus. We have developed quantitative metrics for the evolutionary signatures specific to protein-coding regions and applied them genome-wide, resulting in 1193 candidate new protein-coding exons in the D. melanogaster genome. We have reviewed these predictions by manual curation and validated a subset by directed cDNA screening and sequencing, revealing both new genes and new alternative splice forms of known genes. We also used these evolutionary signatures to evaluate existing gene annotations, resulting in the validation of 87% of genes lacking descriptive names and identifying 414 poorly conserved genes that are likely to be spurious predictions, noncoding, or species-specific genes. Furthermore, our methods suggest a variety of refinements to hundreds of existing gene models, such as modifications to translation start codons and exon splice boundaries. Finally, we performed directed genome-wide searches for unusual protein-coding structures, discovering 149 possible examples of stop codon readthrough, 125 new candidate ORFs of polycistronic mRNAs, and several candidate translational frameshifts. These results affect >10% of annotated fly genes and demonstrate the power of comparative genomics to enhance our understanding of genome organization, even in a model organism as intensively studied as Drosophila melanogaster.

Evidence of abundant stop codon readthrough in Drosophila and other metazoa

“…While evolutionary signatures associated with proteincoding selection usually showed sharp boundaries coinciding with the exact boundaries of protein-coding genes, we found a surprising 149 readthrough candidates for which the protein-coding constraint extends past the stop codon until the next in-frame stop codon ( Fig. 1; Lin et al 2007), suggesting not only that translation does not always stop at the stop codon but also that the specific polypeptide sequence of the extended protein confers selective advantages at the protein level. At the time, we ruled out selenocysteine insertion, but postulated that perhaps adenosine-to-inosine (A-to-I) editing was responsible for the observed signature, by editing away stop codons into tryptophan codons, since readthrough candidates were enriched for nervous system genes where editing is most active.…”

Discovery of functional elements in 12 Drosophila genomes using evolutionary signatures

“…Comparative information has improved computational gene predictors 5 , but their accuracy still falls far short of well-studied gene catalogues such as the FlyBase annotation, which combines computational gene prediction 37 , high-throughput experimental data [38][39][40][41][42] and extensive manual curation 23 . Recognizing this, we set out not only to produce an independent computational annotation of protein-coding genes in the fly genome, but also to assess and refine its already high-quality annotations 43 .…”

“…2b), recent nonsense mutations ( Supplementary Fig. 2c) and alternative translational reading frames 43 .…”

Global analysis of Drosophila Cys2-His2 zinc finger proteins reveals a multitude of novel recognition motifs and binding determinants

“…For genes with multiple well-separated finger clusters, the clusters were characterized as independent recognition units. ZFA clusters were obtained by PCR from cDNA clones of the BDGP DGC Gold and TF collections (Stapleton et al 2002;Lin et al 2007) or D. melanogaster genomic DNA. Each zinc finger cluster was cloned as a C-terminal fusion to the omega subunit of E. coli RNA polymerase in the B1H system.…”