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Essential and recurrent roles for hairpin RNAs in silencing de novo sex chromosome conflict in Drosophila simulans [1]

['Jeffrey Vedanayagam', 'Developmental Biology Program', 'Sloan-Kettering Institute', 'New York', 'United States Of America', 'Marion Herbette', 'Laboratoire De Biologie Et Modélisation De La Cellule', 'École Normale Supérieure De Lyon Cnrs', 'Université Claude Bernard Lyon', 'Lyon']

Date: 2023-06

Meiotic drive loci distort the normally equal segregation of alleles, which benefits their own transmission even in the face of severe fitness costs to their host organism. However, relatively little is known about the molecular identity of meiotic drivers, their strategies of action, and mechanisms that can suppress their activity. Here, we present data from the fruitfly Drosophila simulans that address these questions. We show that a family of de novo, protamine-derived X-linked selfish genes (the Dox gene family) is silenced by a pair of newly emerged hairpin RNA (hpRNA) small interfering RNA (siRNA)-class loci, Nmy and Tmy. In the w[XD1] genetic background, knockout of nmy derepresses Dox and MDox in testes and depletes male progeny, whereas knockout of tmy causes misexpression of PDox genes and renders males sterile. Importantly, genetic interactions between nmy and tmy mutant alleles reveal that Tmy also specifically maintains male progeny for normal sex ratio. We show the Dox loci are functionally polymorphic within D. simulans, such that both nmy-associated sex ratio bias and tmy-associated sterility can be rescued by wild-type X chromosomes bearing natural deletions in different Dox family genes. Finally, using tagged transgenes of Dox and PDox2, we provide the first experimental evidence Dox family genes encode proteins that are strongly derepressed in cognate hpRNA mutants. Altogether, these studies support a model in which protamine-derived drivers and hpRNA suppressors drive repeated cycles of sex chromosome conflict and resolution that shape genome evolution and the genetic control of male gametogenesis.

Funding: These funding agencies supported this research: National Institutes of Health K99-GM137077 to JV; French National Research Agency (ANR-16-CE12-0006-01) to RD and BL; National Institutes of Health R01-GM123194 to CM; National Institutes of Health (R01-GM083300, R01-HD108914, P30-CA008748) and Binational Science Foundation BSF-2015398 to ECL. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2023 Vedanayagam et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Overall, we provide evidence connecting multiple enigmatic meiotic drive systems that appear to involve different members of a single X-linked protamine-derived selfish gene family and are correspondingly suppressed by autosomal hpRNA-siRNA loci. These findings testify to rapid evolutionary dynamics of meiotic drive in the male germline and reveal a critical role of endogenous RNAi to suppress intragenomic conflict.

In this study, we address these questions using new, targeted deletions of the Nmy and Tmy loci in a standardized genetic background (D. simulans w[XD1]). These alleles generate distinct phenotypes: nmy/nmy males sire predominantly female progeny, while tmy/tmy genotypes are completely sterile; neither mutant allele affects viability or female fertility. Nevertheless, genetic interactions between these hpRNA mutants provide support for the notion that Tmy is an endogenous suppressor of sex ratio distortion. We identify key targets of Tmy as newly recognized X-linked paralogs of Dox/MDox, referred to as ParaDox genes (PDox1/2) [ 20 , 21 ]. Transgenic assays demonstrate derepression of Dox and PDox2 proteins in nmy and tmy mutant testes, respectively. Finally, using naturally occurring deletion alleles of Dox family loci, we show that males carrying mutant alleles at both driver and suppressors rescue both fertility and sex ratio phenotypes, underscoring the role of intragenomic conflict in driving the evolution of these sequences.

Despite these recent advances, critical questions regarding both the mechanisms and evolutionary history of sex ratio distortion in D. simulans remain unanswered. First, what is the functional and evolutionary relevance of co-regulation of Dox and MDox by the Nmy and Tmy hpRNAs [ 18 ]? Does the Tmy hpRNAs suppress an X-linked driver other than Dox/MDox, as implied by the original semi-sterile phenotype? Second, is focal deletion of the Tmy hpRNA sufficient to cause the tmy mutant phenotype? In particular, the original tmy introgression lines replaced megabases of Dsim sequences with homologous Dmau regions, and even the minimal tmy mutant region defined by partially overlapping introgression is approximately 80 kb. Moreover, it was proposed that there are complex genetic interactions in the tmy region that affect male fertility [ 17 , 22 , 23 ]. Thus, more precise genetics are required to clarify the extent to which loss of the Tmy hpRNA is responsible for the tmy phenotype defined by inter-species introgressions. Third, what are the functional products of Dox family loci, and how do they preferentially affect Y-bearing sperm? To date, there has not been direct evidence for protein products of Dox family loci, and the only evidence for their derepression in drive situations is at the transcript level [ 18 ].

(A) Biogenesis of hpRNAs into siRNAs via core RNAi factors Dcr-2/AGO2. We used CRISPR/Cas9 and multiple gRNAs to generate Dsim tmy (B-E) and nmy (F-J) deletion alleles for phenotypic analysis. (B) Tmy genomic region showing RNA-seq and small RNA-seq data from w[XD1] in the vicinity of the Tmy hpRNA. We used multiple gRNAs to delete tmy and replace it with a ubi-GFP marker, which is visible throughout the animal (C). (D) Assays of male reproduction reveal complete sterility of tmy[GFP] mutants at 3 different temperatures. (E) Nmy genomic region showing RNA-seq and small RNA-seq data from w[XD1] in the vicinity of the Nmy hpRNA. We used multiple gRNAs to delete nmy and replace it with a 3xP3-DsRed marker (F). (G) Assays of male reproduction reveal nearly complete loss of male progeny by nmy[DsRed] mutants at 18°C. (H) Summary of nmy (“Winters” suppressor) and tmy (“Durham” suppressor) knockout phenotypes in the w[XD1] background. Statistical tests were Wilcoxon rank-sum test of differences between medians. hpRNA, hairpin RNA; siRNA, small interfering RNA.

We previously reported that Nmy encodes hpRNA-class small interfering RNAs (siRNAs) generated by Dcr-2/AGO2, which directly repress Dox and MDox ( Fig 1A ) [ 18 ]. We discovered this network of X-linked distorters and hpRNA suppressors to be more extensive than previously appreciated, by identifying a novel hpRNA in the genetic interval defined by tmy introgressions. The Tmy hpRNA contains regions of 80% to 90% sequence identity with Nmy and is capable of repressing Dox and MDox in trans [ 18 ], indicating a mechanistic link between the Winters and Durham SR systems. Moreover, recent advances in genome assemblies [ 19 ] revealed further expansion and rapid evolutionary divergence of the X-linked Dox gene family and autosomal Nmy/Tmy-related hpRNAs in Dsim, Dmau, and Dsech [ 20 , 21 ]. Together, these findings point to ongoing and rampant sex chromosome conflicts across these 3 simulans-clade species.

Two of these SR systems were discovered through experimental introgression between Dsim and 2 other closely related Drosophila. In the Winters system, introgression of an autosomal Drosophila sechellia (Dsech) locus into the Dsim background revealed a pair of distorter loci on the X chromosome named Distorter on X (Dox) and its paralog Mother of Dox (MDox), which mediate SR drive by an as-yet unknown mechanism [ 15 ]. The autosomal suppressor locus that counteracts Dox to restore balanced sex ratio was mapped to a hairpin RNA (hpRNA) encoding locus named Not much yang (Nmy) [ 16 ]. In the Durham system, autosomal introgression of Drosophila mauritiana (Dmau) sequences into Dsim caused both loss of fertility and female-biased progeny, suggesting another autosomal SR suppressor named Too much yin (Tmy). Tmy was hypothesized to suppress a putative X-linked meiotic drive locus [ 17 ] that remains to be functionally identified.

By biasing transmission of the X or Y, meiotic drive loci located on sex chromosomes generate skewed progeny sex ratio (SR). This makes sex-chromosome meiotic drive much easier to detect than autosomal drive, especially in biological systems without genetic or genomic resources [ 10 ]. However, the molecular identities of many endogenous drive factors and their modus operandi of mediating sex chromosome bias remain mostly mysterious. The insect clade has been particularly effective for revealing SR drive systems, including in numerous Drosophila species, stalk-eyed flies, butterflies, and mosquitoes [ 6 , 10 – 13 ]. Among these, analysis of Drosophila simulans (Dsim) suggested 3 distinct SR drive systems in this individual species [ 5 , 14 ], while none have yet been discovered in the major experimental model D. melanogaster (Dmel), a very close relative of Dsim.

Chromosomal sex determination provides unique evolutionary opportunities for meiotic drive elements that differ from autosomal systems. Unlike most autosomal homologs, sex chromosomes typically harbor highly distinct genetic and sequence content, which enables meiotic drive loci to distinguish the chromosome where they reside from the alternative homolog [ 4 – 6 ]. Across many animal species, parents maximize fitness by investing equally in male and female offspring, and consequently natural populations usually maintain an equilibrium sex ratio determined by the relative costs of producing male versus female offspring [ 7 , 8 ]. If this cost is equal, the equilibrium sex ratio will be 1:1. However, if populations deviate from this equilibrium, selection then favors alleles which produce offspring of the rarer sex, ultimately restoring the population to the Fisherian equilibrium [ 9 ]. These considerations suggest that meiotic drive elements located on sex chromosomes may be countered by unlinked loci across the genome. Mutations on the targeted homolog that escape or suppress the driver, as well as at autosomal loci that restore the Fisherian sex ratio and/or recover male fertility lost from gamete destruction can propagate cycles of coevolution between drivers and suppressors.

Meiosis is a specialized germline cell division that produces haploid gametes from diploid progenitor cells. During meiosis, equal segregation of alleles is critical for faithful transmission of genetic information and for maintenance of euploid genome integrity. While equal segregation is the norm, exceptions can occur in the form of meiotic drive. Meiotic drive results when normal meiosis is compromised by selfish genetic elements, DNA sequences that manipulate meiosis to gain a biased transmission advantage to the next generation. Over the last few decades, meiotic drive systems have been documented across diverse eukaryotes, including plants, fungi, insects, and mammals [ 1 – 3 ]. Thus, the status quo of Mendel’s law of segregation is frequently breached.

Results

Precise deletion of the Tmy Nmy hpRNA yields complete male sterility Over 20 years ago, Yun Tao analyzed introgressions between closely related simulans-clade Drosophila species to identify genomic intervals underlying F1 hybrid male reproductive defects observed in crosses between Dsim and Dmau [17,23,24]. In particular, introgression of Dmau regions on chromosome arm 3R into Dsim revealed the tmy phenotype, which manifests both as subfertility and an excess of female progeny [17]. Although the causal interval harboring tmy defined by overlapping introgressions was localized to <100 kb, no candidate gene was proposed until our recent discovery that an hpRNA class endo-siRNA locus resides in the Tmy region [18]. hpRNAs are initially transcribed as long mRNA-like primary hpRNA transcripts, which are processed by Dcr-2 into approximately 21 to 22 nt siRNAs that program AGO2 effector complexes for gene silencing (Fig 1A). The discovery that the Tmy hpRNA is absent from the introgressed Dmau region [20,21] is consistent with the hypothesis that the original phenotype is due to the loss of Tmy. Still, there was no direct evidence that the Tmy hpRNA maintains fertility and/or sex ratio balance, and unfortunately, the original tmy introgression lines are no longer extant. We used multiplex gRNA strategies to delete the Tmy hpRNA locus and replace it with a ubi-GFP marker (tmy[GFP], Fig 1B) in the parent strain Dsim w[XD1]. We isolated multiple independent founder alleles based on fluorescence (Fig 1C) and genotyped both flanks to verify on-target donor replacements as well as confirm loss of Tmy genomic sequences (S1 Fig). All of these alleles proved viable and female fertile as homozygotes or as trans-heterozygotes; however, all tmy[GFP] mutant genotypes were completely male sterile (Fig 1D). Since spermatogenesis can be temperature dependent, we further tested tmy[GFP] mutants at 17°C, 25°C, and 29°C. These mutants remained sterile under all of these conditions (Fig 1D). Thus, targeted deletion shows that a de novo hpRNA is completely essential for male fertility in the w[XD1] background.

Precise deletion of the Nmy hpRNA yields recapitulates strong sex ratio bias Previous studies of nmy mutant alleles used heterogeneous genetic backgrounds [16,18,25]. Moreover, these nmy alleles are unmarked, rendering genetic crosses technically challenging, especially as this species lacks balancer chromosomes. We therefore deleted the Nmy hpRNA in w[XD1] and replaced it with 3xP3-DsRed (i.e., nmy[DsRed], Fig 1E and 1F), permitting independent visual identification of both hpRNA alleles. We isolated multiple independent founder alleles and again genotyped both flanks to verify on-target donor replacements and confirm loss of Nmy genomic sequences (S1 Fig). All of these alleles proved homozygous viable, but recapitulated previously described male-specific reproductive phenotypes, namely that they are fertile but sire highly female-based progeny (approximately 90% at 18°C, Fig 1G). Thus, both Winters and Durham drive systems are fully active in w[XD1] and can be investigated using our nmy and tmy deletion alleles in a common genetic background (Fig 1H).

Loss of Tmy and Nmy hpRNAs yields distinct cytological defects in testis We performed cytology to assess testis defects that underlie these hpRNA mutant phenotypes. The overall cellular organization and architecture of the Drosophila testis, and details of meiotic divisions, are schematized in Fig 2. During normal spermatogenesis, the mitotic and meiotic products of germline stem cell division remain connected by cytoplasmic bridges, yielding 64 spermatids that undergo coordinated elongation. Staining for histones, F-actin and DAPI reveals the orderly arrangements of actin cones within individualization complexes (ICs) of each meiotic cluster, and the progression of nuclear morphological changes from round in spermatids to needle-shaped in mature spermatozoa (Figs 2 and 3A). Compared to control w[XD1], nmy[DsRed] homozygotes execute gross aspects of spermatogenesis normally, but tmy[GFP] homozygotes exhibit highly disorganized cysts with scattered spermatid nuclei and ICs and they fail to deposit mature sperm in the seminal vesicle (SV) (Fig 3A and 3B). tmy mutants are similar to complete loss of RNAi activity in ago2 mutants, in that both are sterile and fail to complete spermatogenesis, but the latter are more severe and lack ICs altogether (Fig 3A and 3B). PPT PowerPoint slide

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TIFF original image Download: Fig 2. Overviews of the Drosophila testis and male meiotic divisions. (A) Schematic organization of the Drosophila testis. Spermatogenesis begins at the apical tip and progresses along in a spatiotemporal manner. The apical tip contains the hub, which contains a small number of self-renewing germline stem cells (purple). By asymmetric division, the stem cells give rise to gonial cells, which will in turn undergo 4 synchronous mitotic divisions with incomplete cytokinesis, forming a cyst of 16 interconnected primary spermatocytes. After a period of growth, all spermatocytes undergo meiosis I and II in synchrony (detailed in the inset, B) to form 64 interconnected spermatids. The 64 spermatids differentiate synchronously into mature spermatozoa (only 8 are shown for clarity), during which their nuclei undergo a morphological change from round to needle-shaped. This is achieved by replacing histones (red) with transition proteins (orange), and then by SNBPs (i.e., protamines, green), yielding nuclei with a compact and elongated shape. At the same time, each cell produces a flagellum that extends almost the entire length of the testis. At the end of the histone-to-protamine transition, spermatids are individualized by actin cones (blue) that assemble around the spermatids and move coordinately from head to tail, removing excess cytoplasm. The actin cones end up in a waste bag near the apical part of the testis. Finally, mature spermatozoa coil and are released at the proximal end of the testis into the SV. (B) Drosophila male meiosis. Nuclei are outlined, red shading indicates chromatin. For clarity, only 1 primary spermatocyte, out of the 16 present inside a cyst, is shown. Meiosis produces 4 haploid (1N) daughter cells from a diploid primary spermatocyte (2N). After S and G2 phases, the homologous chromosomes pair up. Three territories are formed, separating the non-homologous chromosomes from each other. Two territories are formed by the 2 autosomes (chromosomes II and III) and the third by the X, Y and fourth chromosomes. The chromosomes condense in prophase I and align on the metaphase plate. Then, homologous chromosomes are separated. At the end of meiosis I, 2 daughter cells are formed, which proceed to meiosis II to separate sister chromatids and then give 2 haploid (1N) daughter cells each. SNBP, sperm nuclear basic protein; SV, seminal vesicle. https://doi.org/10.1371/journal.pbio.3002136.g002 PPT PowerPoint slide

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TIFF original image Download: Fig 3. Cytological basis of male reproductive defects in nmy and tmy knockouts. (A, B) Confocal images of whole mount testes from control flies (w[XD1] as wild-type control) and mutants (nmy, tmy, and ago2) stained with a pan-Histone antibody (red) and phalloidin (F-actin green) to reveal ICs and DAPI to label nuclei (blue). (A) Whole testis images, with the stem cell regions labeled with asterisks and seminal vesicles labeled with SV. (A) Control w[XD1] testis shows the orderly progression from mitotic (1), meiotic (2), and differentiation (3) regions. (B) nmy mutants exhibit grossly normal spermatogenesis with normal ICs (arrows, A’). Spermatogenesis is highly disturbed in tmy and ago2 homozygous mutants, with disorganized cysts and aberrant (tmy) or absent (ago2) IC (A’). The empty SVs. Scale bars: 100 μm. (B) Focus on the SV shows they are filled with sperm in control and nmy mutants, as seen with needle-shaped DAPI staining, but are empty in tmy and ago2 mutants; the large round nuclei at the borders correspond to the cells of the somatic wall. (C, D) Cysts of 64 spermatids in control (w[XD1]) and nmy mutants (grown at 18°C). The stages of the histone-to-protamine transition indicated were determined with pan-Histone staining and nuclear shape. Histones are eliminated in both control (C) and nmy (D) spermatids. However, individualization of control spermatid nuclei yields regular needle shapes that stain uniformly with DAPI, whereas about half of nmy mutant nuclei fail to elongate and exhibit abnormal shape (arrow). In addition, DAPI staining in nmy mutant nuclei is uneven, with both less (triangle) or more (DAPI foci, arrowhead) dense regions, suggesting aberrant chromatin organization. Scale bars: 10 μm. (E, F) Meiosis in control and tmy homozygous testes stained for histones (red) and α-tubulin (green). The stages indicated were determined by counting nuclei number in cysts (16 for meiosis I and 32 in meiosis II). In tmy mutants, chromosomes are fragmented and chromatin bridges are observed in anaphase and telophase of both meiosis I and meiosis II. Scale bars: 10 μm. IC, individualization complex; SV, seminal vesicle. https://doi.org/10.1371/journal.pbio.3002136.g003 More detailed examination of mature cysts during the histone-to-protamine transition revealed that histones are removed normally in nmy mutants, but about half of the nuclei fail to elongate and are instead irregularly shaped (Fig 3C and 3D). Such defects remain during individualization, where control spermatid nuclei are needle shaped and stain uniformly with DAPI, but roughly half of nmy mutant nuclei are abnormally shaped and lack uniform chromatin condensation. DNA FISH experiments labeling the sex chromosomes indicate that the affected nmy nuclei correspond to Y-bearing sperm (S2 Fig), consistent with previous reports [25]. By contrast, tmy mutants exhibit severe defects in meiotic nuclei. In particular, histone/α-tubulin/DAPI staining revealed fragmented chromosomes and chromatin bridges in anaphase and telophase of meiosis I and meiosis II (Figs 2E and 2F and S3). Thus, while loss of Nmy causes largely postmeiotic defects, loss of Tmy affects meiotic progression. Overall, these analyses demonstrate that deletion of the Nmy and Tmy hpRNAs result in strong, but distinct, male reproductive phenotypes. This is striking since knockout of de novo genes in Dmel rarely results in detectable phenotypes; although it is notable that when they do, they often impair spermatogenesis [26]. Moreover, the difference between the total sterility of our targeted tmy deletion mutants, and the partial fertility reported in previously described tmy introgression lines [17], suggested that hpRNA knockout defects might be modified by genetic background. We address and confirm this hypothesis later in this study.

Preferred suppression of distinct Dox family targets by Nmy and Tmy hpRNAs We reported that all D. melanogaster hpRNAs exhibit 1 or a few highly complementary targets [27], supporting a scenario by which hpRNAs generally derive from duplications of their target genes. We reported that Tmy is capable of suppressing Dox and MDox in ectopic sensor assays [18]. However, the distinct mutant phenotypes of the 2 Dsim-specific hpRNAs indicate that Tmy likely represses loci beyond Dox and MDox. In a recent study, we searched the highly contiguous D. simulans w[XD1] genome [19] for other sequences homologous to Tmy. The long read assembly is particularly useful for this purpose as repetitive loci are often misassembled using short reads. Indeed, loci in this meiotic drive regulatory network are misassembled in previously available D. simulans genomes [18], because Nmy and Tmy encode related autosomal loci that include highly complementary inverted repeats, which also match the X-linked target genes Dox and MDox. We reported that the uncharacterized X-linked loci GD27797A/B are homologous to Dox and MDox, but exhibit more extensive homology to Tmy compared to Nmy [20]. Accordingly, we named them Paralog of Dox genes (PDox1/2). None of these genes are found in the Dmel genome, but they comprise a large family of loci that are rapidly evolving and proliferating on the X chromosomes of the 3 simulans-clade species [20,21]. Are PDox genes bona fide Tmy targets, and to what extent do Nmy and Tmy exhibit target specificity versus overlapping suppression? To address these questions, we first conducted heterologous gain-of-function assays to confirm these hpRNAs can directly repress Dox genes. We previously published that both Nmy and Tmy hpRNAs can specifically repress Dox and MDox luciferase sensors when overexpressed in a non-cognate setting (D. melanogaster S2 cells) [18]. We constructed PDox1 and PDox2 sensors and tested their response to either wild-type or mutant constructs of Nmy and Tmy. Interestingly, only wild-type Tmy could repress PDox sensors, suggesting that it might be their endogenous suppressor (Fig 4A). PPT PowerPoint slide

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TIFF original image Download: Fig 4. Specific and overlapping roles for Nmy and Tmy hpRNAs in suppressing Dox family targets and male reproduction. (A) Gain-of-function assays of hpRNAs and luciferase targets of PDox1 and PDox2 in D. melanogaster S2 cells. Only wild-type Tmy has capacity to repress both PDox sensors. (B) Loss-of-function assays of testis RNA from D. simulans hpRNA knockouts. qPCR assays show that nmy mutants specifically derepress Dox and MDox, while tmy mutants specifically misexpress PDox genes (amplicons could not distinguish these highly similar transcripts). (C, D) Genetic interactions support an endogenous role for Tmy in sex ratio control. (C) tmy/+ heterozygous fathers exhibit even sex ratios in their progeny, similar to w[XD1] fathers. (D) nmy mutant males are temperature sensitive and their progeny sex ratio bias decreases from 90%–95% at 17°C to modest distortion at 25°C. Removal of 1 allele of tmy clearly enhances nmy SR defects at 22°C and 25°C. (E) Cytological analysis demonstrates that tmy is a dose-sensitive enhancer of nmy during spermatogenesis. Cysts of 64 differentiating spermatid nuclei before and during individualization were stained for histones and DAPI. In control w[XD1] and tmy/+, spermatids elongate normally. At restrictive temperature (18°C), about half of nmy spermatids fail to elongate normally, but this is strongly suppressed at permissive temperature (25°C). Loss of 1 tmy allele in nmy mutants at 25°C phenocopies strong nmy defects seen at 18°C. Scale bar: 10 μm. (F) Summary of specific and cross-regulatory repression of de novo X-linked Dox family genes by autosomal hpRNAs Nmy/Tmy. The raw data for the luciferase sensor assays are provided in S1 Data and raw data for qPCR assays in S2 Data. hpRNA, hairpin RNA. https://doi.org/10.1371/journal.pbio.3002136.g004 We next isolated RNA from testis of w[XD1], nmy[DsRed] homozygotes, and tmy[GFP] heterozygotes and homozygotes, and used qPCR to assess expression of Dox, MDox, and PDox1/2 (due to their high degree of identity, we were not able to distinguish individual PDox loci). We observe notable target specificity in these hpRNA mutants (Fig 4B). Consistent with our prior tests with wild nmy mutant alleles [18], Dox and MDox were both derepressed in our newly generated nmy[DsRed] mutants; however, PDox1/2 remained suppressed. Reciprocally, PDox transcripts were strongly overexpressed in homozygous tmy[GFP] mutants, but Dox and MDox were only subtly derepressed. None of these target genes are overexpressed in tmy[GFP] heterozygotes, indicating that repression by wild-type Tmy hpRNA is dominant. Together, these data support the genetic hypothesis that derepression of distinct Dox gene family members causes the different nmy and tmy mutant phenotypes observed here and in prior introgression studies [16,17].

Dominant genetic interactions reveal an endogenous role of Tmy in sex ratio control The complete sterility of w[XD1]; tmy males precluded direct assessment of Tmy in SR control in this genetic background. However, the Winters drive system is temperature sensitive, such that nmy mutant sex ratio bias is reduced at higher temperature [16]. We tested our new nmy[DsRed] allele and observed that progeny sex ratio bias from nmy[DsRed] homozygous fathers decreased as the temperature increased from 17°C/22°C/25°C (Fig 4C). Accordingly, we sought to test if heterozygosity for tmy[GFP] sensitizes the loss of Nmy at permissive temperatures. We first documented that tmy heterozygous males do not exhibit any sex ratio bias in their progeny, yielding sex ratios that are similar to control w[XD1] males (Fig 4C). Next, to test for genetic interactions between the 2 hpRNA mutants, we took advantage of their independent visual markers. This allowed us to generate a recombinant double mutant chromosome (tmy[GFP], nmy[DsRed]), which we crossed with nmy[DsRed]/nmy[DsRed] to generate male flies that were homozygous for nmy[DsRed] and heterozygous for tmy[GFP]. We observed that tmy dominantly enhanced the sex ratio bias of homozygous nmy mutants at both 22°C and 25°C (Fig 4D), indicating that Tmy is a physiological suppressor of Winters sex ratio drive. We bolstered this conclusion using cytological analysis. At 18°C, nmy mutants show defects in half of the spermatids (presumably Y-bearing), but this phenotype was alleviated at 25°C (Fig 4E), consistent with the temperature effects on sex ratio in this genotype (Fig 4D). However, tmy[GFP], nmy[DsRed]/+, nmy[DsRed] males showed far greater spermatid disruption than nmy mutants, with aberrant nuclei observed at both pre-IC and IC stages (Fig 4E). Overall, these results reveal complexity in the functional roles of Nmy and Tmy. These hpRNAs appear to have distinct primary targets within the Dox family, with Nmy as the primary suppressor of Dox/MDox and Tmy as the major suppressor of PDox1/2 (Fig 4F). However, we have also shown that both Nmy and Tmy are capable of repressing Dox and MDox sensors [18], while only Tmy is further capable of suppressing PDox1 and PDox2 sensors (Fig 4A). Taken together, these observations suggest that Tmy provides subsidiary repression of Dox/MDox, which becomes particularly overt in the nmy mutant context. However, further tests will be needed to distinguish whether PDox factors might also contribute to the phenotype of compound hpRNA mutants.

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