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: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=708132
• 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。以RNA为基础的机制包括逆转录mRNA以产生cDNA.逆转录酶(RT)的来源可能是内源性逆转录转座子。该基因被插入到基因组中的一个新位置，因此缺少其母基因的转录调控元件和内含子。以RNA为基础的基因复制或逆转录基因必须在其插入部位获得启动子才能表达。因此，逆转录酶基因从一开始就不太可能有与其母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，但它们既可以保留其蛋白质编码能力，也可以作为一种调节性的非编码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，我们将重点放在逆转录基因复制上。我们利用果蝇和其他物种可用的大量工具包来解决三个短期目标：1)确定不同谱系中编码蛋白质的逆转录基因及其亲本基因的功能多样性；2)鉴定和确定逆转录复制产生的转录假基因的功能；以及3)确定胚系中逆转录复制的机制。 1)我们研究了三个亲本基因-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。通过比较母基因和逆转录基因在胚胎发育过程中的表达模式，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，并且这些模式在不同物种之间可能会有所不同。相比之下，亲本基因的表达模式是保守的。因此，逆转录基因受到的约束似乎比亲本基因要少。为了建立在这些发现的基础上，我们建议通过集中的发育研究来检查Sep2/Sep5亲本基因-逆转录基因对在黑腹毛虫中的多样性，并在其他物种中使用新的靶向基因敲除技术，使我们能够直接测试功能多样化的进化意义。 2)自从发现了具有调节功能的长非编码RNA以来，假基因的定义发生了变化，例如微RNA结合的诱饵。此外，观察到很大比例的真核基因组在转录上是活跃的，这也导致了人们对“垃圾”DNA具有功能性的猜测。转录的假基因是否具有普遍的功能，这一问题需要通过序列进化和遗传学研究来解决。为了我们的第二个目标，我们将通过在一个亲本基因转录的假基因对上的工作来解决这些问题，以描述其他亲本基因-伪基因对的特征。我们将研究它们在黑腹毛虫和其他假基因序列被保守和转录的物种中的功能。 3)在生殖系中逆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式mRNA模板中产生一个cdna插入。然而，逆转录转座子沉默piRNA途径控制着它们在生殖系中的活性。这一途径的缺失会导致不育，这可能是由于逆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回溯复制的机制。我们将通过两种方法调查在发育不良的果蝇中反转录的mRNAs的类型和频率：(A)通过表位标记、免疫沉淀和RNA测序分离与逆转座子蛋白结合的RNA和(B)在DNA断裂处测序cDNAs。