Alternative routes to optimal expression levels: Evolutionary evidence for competitive RNAs and dosage compensation by gene duplication

Abstract

Expression evolution and dosage constraints are important factors shaping genomic content and innovation. It has become increasingly clear that non-coding RNAs perform various regulatory functions in different cellular processes. The competitive endogenous RNA (ceRNA) hypothesis is an attractively simple model to explain the biological role of many putatively functionless non-coding RNAs. Under this model, there exist transcripts in the cell whose role is to titrate out miRNAs such that the expression level of another target sequence is altered. Multiple examples of pathogenic effects of inappropriate expression of MRE-containing RNAs suggest that this is logistically possible. However, the role, if any, of ceRNAs in normal biological processes and at physiological levels is disputed. By comparison of parent genes and pseudogenes we show, both for a specific example and genome-wide, that the pseudo-3?UTRs of expressed pseudogenes are frequently retained and are under selective constraint in mammalian genomes. We find that the pseudo-3?UTR of BRAFP1, a previously described oncogenic ceRNA, has reduced substitutions relative to its pseudo-CDS, and we show sequence constraint on MREs shared between the parent gene, BRAF, and the pseudogene. Investigation of RNA-seq data reveals expression of BRAFP1 in normal somatic tissues in human and in other primates, consistent with biological ceRNA functionality of this pseudogene in non-pathogenic cellular contexts. On a genome-wide scale we found that pseudo-3?UTRs of mammalian pseudogenes are under stronger selective constraint than their pseudo-CDS counterparts, and are more often retained and expressed. Our results suggest that many human pseudogenes, often considered non-functional, may have an evolutionarily constrained role, consistent with the ceRNA hypothesis. Evolution of therian sex chromosomes is an interesting case of how dosage constraints have shaped gene content on the X chromosome, requiring the resolution of dosage imbalances through several mechanisms, including upregulation of X-linked genes and Y gametolog retention. Here we examined an alternative hypothesis where high maximally expressed genes on the X duplicated in tandem to resolve dosage imbalances due to transcriptional 'traffic jams'. We found an increase in tandem duplication of constrained X-linked genes after sex chromosome formation, but young (post-X) duplicates are not enriched for high maximally expressed genes. Our observations do not support tandem duplication on the X as a means of resolving dosage imbalances due to transcriptional haploidy, but may suggest an alternate route to dosage balance for copy number constrained dosage-sensitive genes