Fact or fiction: updates on how protein-coding genes might emerge de novo from previously non-coding DNA

Identification of de novo genes

The first step necessary to determine de novo status of a gene is to verify that no homologous sequences are present in outgroups. This homology search is often performed using BLAST or similar alignment search tools, for example against non-redundant protein databases containing all known protein sequences. Usually, an e-value cutoff between 10⁻³ to 10⁻⁵ is used for this step to ensure that no spurious, suboptimal alignments are taken into account⁴. If this homology search does not find any homologs outside of the analyzed species, the query gene has successfully been confirmed to be a novel gene. This definition states only that there are no homologous sequences outside of a certain phylogenetic group. Calling a gene novel does not imply any knowledge about the emergence mechanism of the gene.

To additionally determine de novo gene origin, the homologous non-coding outgroup DNA sequence has to be retrieved^14,23. The outgroup homologous sequence can be recovered using synteny information about the position of orthologous neighbor genes. Another possibility is searching the target gene sequence in outgroup genomes using alignment search tools such as BLAST^4,23. A number of different types of de novo genes can be discriminated depending on the type of sequence that the genes likely emerged from²³.

Problems in de novo gene identification and annotation. In the past²⁴ and also more recently^25,26, studies have raised questions regarding the reliability of homology-based searches of novel genes. Specifically, short and fast-evolving genes were proposed to lose detectable sequence similarity faster than other genes. As a result, shorter genes would be expected to be over-represented among young genes, thereby biasing the results of studies of genes of different ages^24–26. Doubts have been raised as to which fraction of genes would actually be affected by this effect²⁷. Also, this should not be a problem for de novo genes defined by the methods summarized here. The possibility that the examined gene is actually a fast-evolving old gene is excluded, since for a confirmed de novo gene the homologous non-genic outgroup sequence has to be determined. Additionally, doubts have been raised regarding the accuracy of the initial claims of the unreliability of homology detection²⁸.

Another challenge is the previously mentioned identification of a non-coding sequence in an outgroup which is clearly homologous to the suspected de novo gene. In non-coding DNA, homology signals disappear very quickly, since non-coding sequences accumulate mutations faster than coding sequences. Because of this, it is often impossible to determine the homologous non-coding sequence in an outgroup. This problem increases with gene age. As a result, it is often not possible to determine the mechanism of origin, especially for older genes.

Additionally, there are methodological difficulties in the annotation of de novo and also all other types of novel genes⁴. These problems could lead to a systematic underestimation of the number of de novo/novel genes. The problems are caused by genome annotation also being based on sequence homology²⁹. As de novo/novel proteins per definition do not possess any homologs, they cannot be annotated based on that criterion and their number is likely to be underestimated. Other common criteria such as minimum expression strength and the presence of multiple exons could also contribute to the problem, as these criteria do not represent intrinsic requirements for gene existence and are biased against de novo/novel genes¹⁸. Nevertheless, the criteria might be necessary to prevent an over-annotation of spurious transcripts as genes, but they also make it impossible to identify all de novo genes. Recent studies on de novo protein-coding genes also employed such thresholds on exon number and expression strength to produce a more robust data set^15,17,18.

De novo gene emergence

Conceptually, de novo genes can evolve via two different mechanisms. The first mechanism is transcription-first, where an intergenic sequence gains transcription before evolving an ORF^20,30. Recently, this has been shown to happen frequently when long non-coding RNAs (lncRNAs) become protein coding^17,31,32. Consequently, lncRNAs could represent an intermediate step in the evolution of a protein-coding gene³³. The second model is ORF-first, in which an intergenic ORF gains transcription^20,30. Such a transcribed de novo ORF has been proposed to represent an intermediate step in gene emergence, a protogene (Figure 1). High turnover of intergenic transcription³⁴ likely plays a role in de novo gene emergence by exposing novel transcripts to selection. Transposable elements can also play a role in de novo gene emergence³⁵. Additionally to whole proteins, terminal domains can also emerge de novo^33,36. One model regarding the emergence of novel domains is the “grow slow and molt”, in which reading frames get extended gradually and eventually gain a structure and function^37,38.

An additional process that could play a role during de novo protein-coding gene emergence is a (partial) revival of pseudogenized gene fragments. This possibility has already been proposed by Ohno¹. Regarding de novo protein-coding gene emergence, it seems possible that fragments of a pseudogenized gene that has been somewhat eroded by drift could become part of a de novo ORF later on. These fragments could provide a starting point for de novo protein emergence by providing remnants of structural elements. For all of these models, there are several consistent findings, but none of the models is, as yet, supported by a comprehensive set of data from diverse sources and corresponding experimental data.

De novo gene death. Orphan genes seem to generally have a high loss probability^14,39 that seems to be negatively correlated with gene age^40,41. The cause of this correlation is not yet well understood. It seems possible that young orphan genes have not yet gained a function or do not perform transient functions. It is also not clear yet how much of these findings can be transferred to de novo genes, as the studies on this topic examined all novel genes of different emergence mechanisms jointly.

De novo gene functions

A number of studies have examined the functions of orphan genes, some of which may represent de novo-emerged genes. Findings on orphan gene functions include involvement of orphan genes in the stress response^21,42, rapid adaptation to changing environments as well as species-specific adaptations^43,44, and limb regeneration⁴⁵. Additionally, novel genes were found to quickly gain interaction partners and become essential^39,46.

Fewer studies, however, have examined the functions of systematically verified de novo-emerged genes. Generally, a high number of de novo genes was found to be expressed specifically in the testes, at least in Drosophila species^5,6 and primates¹⁸, as well as in plant pollen^16,47. In the mouse, a de novo-emerged RNA gene was found to raise reproductive fitness²². Another study found de novo genes to play a role in the Arabidopsis stress response¹². More specifically, one de novo ORF was found to play a role in male reproduction in Drosophila⁴⁸. Reinhardt et al.⁴⁸ also presented findings suggesting a role of de novo genes in developmental stages of Drosophila. However, these findings have to be interpreted carefully, as the RNAi method used has been shown to produce unreliable results^49,50. A few other examples of functional de novo genes have been found³⁰, while others were not able to determine specific functions of identified de novo-emerged genes¹⁵. The available data suggest that de novo-evolved genes can play a role in many different processes from reproduction to the stress response.

Recently, one study analyzed the function of two putative de novo protein-coding genes in Drosophila melanogaster⁵¹. The two analyzed genes were found to be essential for male reproduction and to have testis-biased expression. Both genes are located inside introns of other, older genes with homologs in outgroups. However, the de novo origin of the analyzed genes could not be confirmed with certainty owing to the outgroup homologous sequences not being identifiable (see above for a general description of this problem).

Protein structure of de novo proteins

Little is known about the protein structures of de novo proteins. Some studies have found a high amount of intrinsic protein disorder⁵² in very young genes^15,51,53, while others have not²¹. A priori, it seems unlikely that de novo-emerging proteins have a well-defined protein structure. Intuitively, it seems more likely for random sequences to be intrinsically disordered instead (see Figure 1). Nevertheless, disordered regions can also be highly functional^52,54 and could as such also represent an evolved state.

Also, contrary to intuition, at least semi-random (restricted alphabet) proteins appear to sometimes have a defined secondary structure^55,56. Additionally, the existing protein structure families appear to have multiple origins⁵⁷. This finding suggests that the emergence of new protein structures is at least possible. Avoidance of misfolding and aggregation, on the other hand, have been proposed to be driving forces of protein evolution^58,59. This observation and the existence of de novo protein-coding genes suggest that de novo proteins have the potential to exhibit a defined structure.

Open questions regarding de novo genes

Despite many advances in recent years, many open questions remain regarding de novo protein-coding genes. One understudied field is the functional characterization of protein-coding de novo-emerged genes. One non-coding RNA gene has been found to have a role in reproduction in the mouse²², and additionally one likely protein-coding gene has been found to be essential for reproduction in Drosophila⁴⁸. However, beyond that, there is a substantial lack of data. Consequently, it remains unclear how de novo protein-coding genes gain their function and if there are some roles that they are more or less likely to carry out.

As described above, the structural characterization of de novo protein-coding genes is still an open question. Previously, ambiguous signals have been found regarding the role of intrinsic disorder in de novo-emerging protein-coding genes^15,21. It would be important to experimentally verify the structure — or lack thereof — of de novo protein-coding genes. Here it is of major interest to determine the proportion of intergenic ORFs with folding potential and also what the implications are for the retention of such ORFs. This would allow further conclusions about de novo gene emergence: if most intergenic, random ORFs are foldable, function would seem to be the bottleneck of de novo protein-coding gene retention. On the other hand, if most confirmed de novo genes are folding, but most intergenic ORFs do not possess folding potential, folding potential would be a bottleneck of de novo protein-coding gene emergence and retention.

Another unsolved problem is how to find specific annotation thresholds for orphans/de novo genes⁴. As described above, a number of their properties make de novo genes difficult to annotate and to be distinguished from transcriptional noise. One solution would be to generate high-quality proteome data using e.g. mass spectrometry. However, this process is still highly expensive and might also not be able to generate a complete picture, since low-frequency peptides are hard to detect⁶⁰. Another method is ribosome profiling, which uses ribosome occupancy of sequences as a measure of translation. This method has been successfully used to show that some transcripts that were previously classified as non-coding could in fact be translated⁶¹.

Additionally, patterns of selection, e.g. measured in the ratio of non-synonymous to synonymous mutations, can be used to infer the coding status of sequences. Genes with a higher fraction of synonymous mutations compared to non-synonymous mutations can be expected to be protein coding and under purifying selection^17,20. However, these measures require a number of orthologs to be present, which makes them of limited use for novel genes. Another possibility is the use of population data for the same purpose, which circumvents the problem of the unavailability of orthologs for novel genes.

As it stands, studies mostly have to rely on arbitrary cutoffs^15,17 and thus might miss a number of genes. It would be of major interest to be able to differentiate de novo genes and protogenes from transcriptional noise. Recent research has already shown that small ORFs (smORFs) can play a functional role^62,63, and consequently it seems quite likely that also very short novel ORFs could be functional. This question also touches upon the problem of differentiating lncRNAs from protein-coding genes, which is often performed via an ORF length cutoff^17,32.

Going forward, it is of major interest to fully characterize a large number of de novo genes in terms of evolutionary, functional, and structural history to be able to draw some general conclusion about their evolution. Specifically, it is of major interest to determine whether a functional role is an exception for protogenes or if most expressed ORFs have a functional impact which mostly does not affect the fitness of the organism at a significant level. If most expressed ORFs have only a negligible fitness effect, they would mostly evolve via drift. Two closely related questions are how and when de novo proteins gain their function: are de novo genes usually functional from the time point of their emergence, or do they gain a cellular task only after a period of drift?