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.
Our genomes are frequently in conflict with selfish genes: genes that increase their transmission through spermatogenesis by killing sperm. In some cases, these selfish genes exploit the RNA interference machinery, which is meant to defend our genomes against invaders. Determining how these selfish genes kill sperm to increase their frequencies will provide a unique perspective on spermatogenesis and help us understand the role of RNA interference in the male germline.
| Larracuente, Amanda M; Ferree, Patrick M (2015) Simple method for fluorescence DNA in situ hybridization to squashed chromosomes. J Vis Exp :52288 |
| Unckless, Robert L; Larracuente, Amanda M; Clark, Andrew G (2015) Sex-ratio meiotic drive and Y-linked resistance in Drosophila affinis. Genetics 199:831-40 |
| Larracuente, Amanda M (2014) The organization and evolution of the Responder satellite in species of the Drosophila melanogaster group: dynamic evolution of a target of meiotic drive. BMC Evol Biol 14:233 |
