Genome evolution through RNA-based gene duplication

基因复制有助于遗传变异和新奇的进化。 基于RNA的机制涉及mRNA的逆转录以产生cDNA。 逆转录酶（RT）的来源可能是内源性逆转录转座子。 cDNA插入基因组中的新位点，因此缺失其亲本基因的转录调控元件和内含子。 为了表达，基于RNA的基因复制或逆转录基因必须在其插入位点获得启动子。 因此，逆转录基因不太可能从一开始就具有与其亲本基因相似的表达模式。 虽然许多逆转录基因变成了无功能的假基因，但它们既可以保留蛋白质编码能力，也可以作为调控性非编码RNA。我们的长期目标是确定基因复制的功能和进化意义。 在这个拟议的研究计划中，我们专注于逆转录基因复制。 我们利用黑腹果蝇和其他物种的大量工具包来解决三个短期目标：1）确定不同谱系中蛋白质编码逆转录基因及其亲本基因的功能多样性; 2）识别和确定由逆转录复制产生的转录假基因的功能; 3）确定生殖系中逆转录复制的机制。**1）我们研究了D.黑腹果蝇和其他果蝇物种。 通过比较亲本基因和逆转录基因在胚胎发生过程中的表达模式，我们发现每个逆转录基因具有与亲本基因不同的表达模式，并且这些模式可以在物种之间变化。 相反，亲本基因的表达模式是保守的。 因此，似乎逆转录基因比亲本基因受到更少的限制。 基于这些发现，我们建议研究D.通过集中的发育研究和在其他物种中使用新的靶向基因敲除技术，我们可以直接测试功能多样化的进化意义。2)假基因的定义已经改变，因为长的非编码RNA的发现服务的调节功能，如诱饵microRNA结合。 此外，观察到大部分真核生物基因组是转录活性的，这导致了“垃圾”DNA是功能性的推测。 转录的假基因是否具有普遍功能的问题需要通过序列进化和遗传学研究来解决。 对于我们的第二个目标，我们将解决这些问题的基础上，我们的工作对一个亲本基因转录的假基因对其他亲本基因pseudoretrogene对的表征。 我们将在D中检查它们的功能。黑腹果蝇和其他物种，其中假基因序列是保守的和转录的。3)生殖系中的逆转录复制机制尚不清楚，但在人类细胞和酵母中的工作表明，逆转录转座子蛋白可以反式从mRNA模板产生cDNA插入。然而，逆转录转座子沉默皮尔纳途径控制它们在生殖系中的活性。 该途径的缺失导致不育，推测是由于反转录转座对基因组完整性的影响。 在这方面，我们的第三个目标是解决逆向重复机制问题。 我们将通过两种方法调查基因缺陷果蝇中逆转录的mRNA的类型和频率：（a）使用表位标记、免疫沉淀和RNA测序分离与逆转录转座子蛋白复合的RNA;（B）通过DNA断裂处的cDNA测序。

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.