Genome evolution through RNA-based gene duplication

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Gene duplication contributes to the evolution of genetic variation and novelty. RNA-based mechanisms involve reverse transcription of mRNA to generate cDNA. The source of reverse transcriptase (RT) is likely endogenous retrotransposons. The cDNA is inserted at a new site in the genome and thus is missing transcriptional regulatory elements and introns of its parent gene. To be expressed, RNA-based gene duplications, or retrogenes, must obtain a promoter at their insertion site. Therefore, retrogenes are unlikely to have expression patterns similar to their parent gene from their outset. Although many retrogenes become functionless pseudogenes, they can either retain their protein coding capacity or act as a regulatory non-coding RNA. Our long-term objective is to determine the functional and evolutionary significance of gene duplications. In this proposed research program, we are focusing on retrogene duplications. We take advantage of the vast toolkit available for Drosophila melanogaster and other species to address three short-term objectives: 1) to determine the functional diversification of protein-coding retrogenes and their parent genes in different lineages; 2) to identify and determine the function of transcribed pseudogenes that arose by retroduplication; and 3) to determine the mechanism of retroduplication in the germ-line. 1) We examined the expression and sequence evolution of three parent gene-retrogene pairs in D. melanogaster and other Drosophila species. By comparing parent gene and retrogene expression patterns during embryogenesis, we found that each retrogene has a different expression pattern than the parent gene, and these patterns can vary among species. In contrast, the parent genes' expression patterns are conserved. Thus, it appears that the retrogenes are under less constraint than parent genes. To build on these findings, we propose to examine diversification of the Sep2/Sep5 parent gene-retrogene pair in D. melanogaster by focused developmental studies and in other species using new targeted gene knockout technology, allowing us to directly test the evolutionary significance of the functional diversification. 2) The definition of a pseudogene has changed since the discovery of long non-coding RNAs serving a regulatory function such as a decoy for microRNA binding. Also, the observation that large proportions of eukaryotic genomes are transcriptionally active has led to the speculation that "junk" DNA is functional. The question as to whether transcribed pseudogenes are generally functional needs to be resolved through sequence evolution and genetic studies. For our 2nd objective, we will address these issues by building on our work on one parent gene-transcribed pseudogene pair to characterization of other parent gene-pseudoretrogene pairs. We will examine their function in D. melanogaster and other species where the pseudogene sequences are conserved and transcribed. 3) The mechanism of retroduplication in the germ line is unknown, but work in human cells and yeast shows that retrotransposon proteins can produce a cDNA insertion from an mRNA template in trans. However, the retrotransposon silencing piRNA pathway controls their activity in the germ line. Loss of this pathway leads to sterility, presumably due to the effect of retrotransposition on integrity of the genome. In this context, our 3rd objective is to address the mechanism of retroduplication. We will survey the types and frequencies of mRNAs that get reverse transcribed in dysgenic flies in 2 ways by (a) isolating RNA complexed with retrotransposon protein using epitope tagging, immunoprecipitation, and RNA sequencing and (b) by sequencing cDNA at DNA breaks.