Orphan genes, lacking detectable homologs in outgroup species, typically represent 10-30% of eukaryotic genomes. Efforts to find the source of these young genes indicate that de novo emergence from non-coding DNA may in part explain their prevalence. Here, we investigate the roots of orphan gene emergence in the Drosophila genus. Across the annotated proteomes of twelve species, we find 6297 orphan genes within 4953 taxon-specific clusters of orthologs. By inferring the ancestral DNA as non-coding for between 550 and 2467 (8.7-39.2%) of these genes, we describe for the first time how de novo emergence contributes to the abundance of clade-specific Drosophila genes. In support of them having functional roles, we show that de novo genes have robust expression and translational support. However, the distinct nucleotide sequences of de novo genes, which have characteristics intermediate between intergenic regions and conserved genes, reflect their recent birth from non-coding DNA. We find that de novo genes encode more disordered proteins than both older genes and intergenic regions. Together, our results suggest that gene emergence from non-coding DNA provides an abundant source of material for the evolution of new proteins. Following gene birth, gradual evolution over large evolutionary timescales moulds sequence properties towards those of conserved genes, resulting in a continuum of properties whose starting points depend on the nucleotide sequences of an initial pool of novel genes.

Comparative genomic studies have repeatedly shown that new protein-coding genes can emerge de novo from noncoding DNA. Still unknown is how and when the structures of encoded de novo proteins emerge and evolve. Combining biochemical, genetic and evolutionary analyses, we elucidate the function and structure of goddard, a gene which appears to have evolved de novo at least 50 million years ago within the Drosophila genus. Previous studies found that goddard is required for male fertility. Here, we show that Goddard protein localizes to elongating sperm axonemes and that in its absence, elongated spermatids fail to undergo individualization. Combining modelling, NMR and circular dichroism (CD) data, we show that Goddard protein contains a large central α-helix, but is otherwise partially disordered. We find similar results for Goddard’s orthologs from divergent fly species and their reconstructed ancestral sequences. Accordingly, Goddard’s structure appears to have been maintained with only minor changes over millions of years.

Insertions and deletions (InDels) are frequently observed in natural protein evolution, yet their potential remains untapped in laboratory evolution. Here we introduce a transposonbased mutagenesis approach (TRIAD) to generate libraries of random variants with short inframe InDels, and screen TRIAD libraries to evolve a promiscuous arylesterase activity in a phosphotriesterase. The evolution exhibits features that differ from previous point mutagenesis campaigns: while the average activity of TRIAD variants is more compromised, a larger proportion has successfully adapted for the activity. Different functional profiles emerge: (i) both strong and weak trade-off between activities are observed; (ii) trade-off is more severe (20-to 35-fold increased k cat /K M in arylesterase with 60-400-fold decreases in phosphotriesterase activity) and (iii) improvements are present in k cat rather than just in K M , suggesting adaptive solutions. These distinct features make TRIAD an alternative to widely used point mutagenesis, accessing functional innovations and traversing unexplored fitness landscape regions.

De novo gene emergence provides a route for new proteins to be formed from previously non-coding DNA. Proteins born in this way are considered random sequences, and typically assumed to lack defined structure. While it remains unclear how likely a de novo protein is to assume a soluble and stable tertiary structure, intersecting evidence from random-sequence and de novo-designed proteins suggests that native-like biophysical properties are abundant in sequence space. Taking putative de novo proteins identified in human and fly, we experimentally characterise a library of these sequences to assess their solubility and structure propensity. We compare this library to a set of synthetic random proteins with no evolutionary history. Bioinformatic prediction suggests that de novo proteins may have remarkably similar distributions of biophysical properties to unevolved random sequences of a given length and amino acid composition. However, upon expression in vitro, de novo proteins exhibit higher solubility which is further induced by the DnaK chaperone system. We suggest that while synthetic random sequences are a useful proxy for de novo proteins in terms of structure propensity, de novo proteins may be better integrated in the cellular system given their higher solubility.