Genome evolution through RNA-based gene duplication

通过基于 RNA 的基因复制进行基因组进化

• 批准号: RGPIN-2014-03890
• 负责人: Clark, Denise
• 金额: $ 2.55万
• 依托单位: University of New Brunswick
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2017
• 资助国家: 加拿大
• 起止时间: 2017-01-01 至 2018-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=628960
• 关键词: Genome; evolution; through; RNA; based

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.

基因复制有助于遗传变异和新颖性的进化。基于rna的机制包括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。该cDNA插入到基因组的新位点，因此缺失了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，逆转录基因从一开始就不可能具有与其亲本基因相似的表达模式。虽然许多逆转录基因成为无功能的假基因，但它们可以保留其蛋白质编码能力或作为调节性非编码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，我们的重点是逆转录基因复制。我们利用黑腹果蝇和其他物种的大量可用工具箱来解决三个短期目标：1)确定蛋白质编码逆转录基因及其亲本基因在不同谱系中的功能多样性；2)鉴定和确定由反转录产生的转录伪基因的功能；3)确定生殖系中逆转录的机制。1)研究了3对亲本基因-逆转录基因对在黑腹果蝇和其他果蝇中的表达和序列进化。通过比较胚胎发生过程中亲本基因和逆转录基因的表达模式，我们发现每个逆转录基因都有不同于亲本基因的表达模式，并且这些模式在物种之间存在差异。相比之下，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的约束更小。基于这些发现，我们建议通过集中的发育研究和新的靶向基因敲除技术，在其他物种中研究黑腹龙Sep2/Sep5亲本基因-逆转录基因对的多样化，从而使我们能够直接测试功能多样化的进化意义。2)自从发现具有调控功能的长链非编码rna（如microRNA结合的诱饵）以来，假基因的定义发生了变化。此外，观察到大部分真核生物基因组具有转录活性，这导致了“垃圾”DNA具有功能的猜测。关于转录的假基因是否具有普遍的功能，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，我们将通过建立我们对一个亲本基因转录伪基因对的工作来描述其他亲本基因伪逆转录基因对来解决这些问题。我们将研究它们在D. melanogaster和其他物种中的功能，其中假基因序列是保守和转录的。3)生殖系中反转录复制的机制尚不清楚，但在人类细胞和酵母中的研究表明，反转录转座子蛋白可以从反转录的mRNA模板中产生cDNA插入。然而，逆转录转座子沉默piRNA途径控制它们在种系中的活性。这一途径的缺失导致不育，可能是由于逆转录对基因组完整性的影响。在这种情况下，我们的第三个目标是解决逆转录的机制。我们将通过以下两种方式调查在基因缺陷果蝇中获得逆转录的mrna的类型和频率：(a)使用表位标记、免疫沉淀和RNA测序分离RNA与反转录转座子蛋白的复合物；(b)在DNA断裂处测序cDNA。