The role of satellite rasiRNA in the Segregation Distorter system of Drosophila

描述(申请人提供)：基因组经常与自私的基因发生冲突，自私基因在一种称为减数分裂驱动的过程中将其传递给下一代，通常是在 有机体的成本。分离错配(SD)是黑腹金龟减数分裂的驱动系统，在SD/SD+杂合子雄性中，SD染色体通过杀死携带敏感等位基因的精子(RSP-2染色体着丝粒附近的卫星重复)传递给95%的后代。SD背后的机制尚不清楚，但它可能涉及RNA干扰(RNAi)，类似于其他减数分裂驱动系统。如果不引用遗传冲突的物种历史(例如，RNAi，快速基因进化)，真核生物精子发生的许多特征就无法解释。了解自私基因，如SD，是如何利用RNAi杀死精子的，将为了解小RNA在精子发生中的作用提供独特的见解。本研究的目的是首先确定SD/SD+杂合子SD+精子细胞功能障碍的原因，其次描述RSP重复相关短干扰RNA在睾丸中的分布，并测试它们在SD/SD+杂合子中是否被破坏。第三，这项提议将检验SD通过干扰睾丸减数分裂后rasiRNA的产生来杀死携带SD+的精子细胞的假设。在SD存在的情况下，具有对扭曲敏感的RSP基因(RSP)的SD+精子细胞在精子细胞将其组蛋白交换为精子特有的精蛋白时，无法浓缩其染色质，以帮助浓缩细胞核，因为它被重塑为精子头。为了确定哪些染色质成分在SD/SD+睾丸中表现出异常定位，该建议将对核心组蛋白进行免疫定位，并分析GFP标记的过渡蛋白、鱼精蛋白和也参与核重塑的鱼精蛋白相关染色质成分的表达(目标1)。本研究的初步结果表明，RSP rasiRNA在减数分裂后在精巢中表达。这项建议将通过荧光原位杂交(AIM 2)寻找SD/SD+睾丸中RSP rasiRNAs的中断来检验SD干扰RSP rasiRNA定位或表达的假设。这一提议还将检验RSP rasiRNAs起源于卫星重复自身之外的基因组位置的假设，正如初步结果所表明的那样。这将通过产生包含在卫星基因外发现的RSP重复的基因组区域的缺失来实现。这些缺失将用于测试RSP rasiRNAs的中断 因此，对携带RSPs的染色体的扭曲(目标2)。最后，这项提议将检验SD干扰睾丸减数分裂后rasiRNA产生的假设。为此，我将使用Illumina短读技术分别对分离的SD/SD、SD/SD+和SD+/SD+睾丸有丝分裂细胞和减数分裂后细胞的小RNA进行测序和比较。如果SD影响RSP rasiRNA的产生，那么SD基因型将与RSP rasiRNA在减数分裂后睾丸解剖中的丰度相关。

DESCRIPTION (provided by applicant): Genomes are frequently in conflict with selfish genes that bias their transmission to the next generation in a process called meiotic drive, often at the cost of the organism. Segregation Distorter (SD) is a well-studied meiotic drive system in D. melanogaster: in SD/SD+ heterozygous males, the SD chromosome is transmitted to 95% of the progeny by killing sperm carrying sensitive alleles of its target locus, Responder (Rsp-a satellite repeat near the centromere of chromosome 2). The mechanism behind SD is unknown but it may involve RNA interference (RNAi), similar to other meiotic driver systems. Many features of spermatogenesis in eukaryotes cannot be explained without invoking a species history of genetic conflict (e.g. RNAi, rapid gene evolution). Understanding how selfish genes, like Sd, exploit RNAi to kill sperm will offer unique insight into the role of small RNAs in spermatogenesis. This proposal aims first to determine the cause of SD+ spermatid dysfunction in SD/SD+ heterozygotes, and second, to describe the distribution of Rsp repeat-associated short interfering RNAs (rasiRNAs) in the testis and test if they are disrupted in SD/SD+ heterozygotes. Third, this proposal will test the hypothesis that SD kills SD+-bearing spermatids by interfering with postmeiotic rasiRNA production in the testis. In the presence of SD, SD+ spermatids with a Rsp locus sensitive to distortion (Rsps), fail to condense their chromatin at a time when spermatids swap their histones for sperm-specific protamines to aid in condensing the nucleus as it is re- shaped into the sperm head. To determine which chromatin components show aberrant localization in SD/SD+ testes, this proposal will immunolocalize core histones and analyze the expression of GFP-tagged transition proteins, protamines and a protamine-associated chromatin component also involved in nuclear re-shaping (Aim 1). Preliminary results described in this proposal show Rsp rasiRNA expression in the testis after meiosis. This proposal will test the hypothesis that SD interferes with Rsp rasiRNA localization or expression by looking for a disruption of Rsp rasiRNAs in SD/SD+ testes using Fluorescence In Situ Hybridization (Aim 2). This proposal will also test the hypothesis that Rsp rasiRNAs originate from a genomic location outside of the satellite repeat itself, as preliminary results suggest. Thi will be accomplished by generating deletions of genomic regions containing Rsp repeats found outside of the satellite locus. The deletions will be used to test for a disruption of Rsp rasiRNAs and consequently, distortion against the Rsps-bearing chromosome (Aim 2). Finally, this proposal will test the hypothesis that SD interferes with postmeiotic rasiRNA production in the testis. To do this, I will sequence and compare small RNAs from dissections of SD/SD, SD/SD+ and SD+/SD+ testes enriched for mitotic and postmeiotic cells, separately, with Illumina short read technology (Aim 3). If SD affects Rsp rasiRNA production, then SD genotype will correlate with Rsp rasiRNA abundance in postmeiotic testis dissections.