Small RNA-mediated suppression of sex chromosome meiotic conflicts during Drosophila male gametogenesis

In sexually reproducing organisms, meiosis is a process by which haploid gametes (e.g. egg and sperm cells) are produced from diploid parental cells [1]. One of the hallmarks of meiosis is the equal segregation of genes/alleles [2]. Meiotic drive is a form of genetic cheating that violates this norm, resulting in skewed transmission of alleles [3]. Indeed, this phenomenon is widespread and has been reported in yeasts [4], many different arthropods [5,6], plants [7], mice [8,9], and at least one instance in humans [10]. The extent to which such violations of Mendelian segregation occur in different organisms remains unknown, as meiotic drive may be unrecognized in the absence of overt phenotypes; however, sex chromosome meiotic drive can readily be observed as a skewed male-to-female ratio among progeny [5]. Although sex chromosome drive has been documented in numerous species, the genes responsible for the drive and the host’s suppression strategies often remain unidentified [11].

In recent decades, research in Drosophila has significantly advanced our understanding of sex chromosome drive and its suppression during male gametogenesis. The Stellate (Ste) locus in Drosophila melanogaster is thought to be a relic meiotic drive system, derived from the amplification of the testis-specific β subunit of protein kinase, CkIIβ2 on the X and Y chromosomes [12-15]. The suppressor of Stellate [Su(Ste)] on the Y chromosome silences X-linked Ste copies via post-transcriptional gene silencing, primarily employing the piwi-interacting RNA (piRNA) pathway, which plays a crucial role in defending against transposable elements (TEs) in the animal germline [16,17]. The genetic characterization of Ste locus in D. melanogaster thus provided the first evidence linking small RNA-mediated silencing of endogenous selfish genes involved in meiotic conflicts between sex chromosomes.

In Drosophila simulans, a sibling species of D. melanogaster, several X-linked drive systems that are absent in D. melanogaster have been identified, highlighting the dynamic evolution of sex chromosome conflicts between closely related species. Interestingly, genetic and molecular characterization of these X-linked drive systems—namely, Paris [18], Durham [19], and Winters [20,21]—have identified the surprising roles of small RNA pathway components in meiotic drive and suppression. In the Paris system, a young and rapidly evolving gene called HP1D2 functions as a meiotic driver to prevent proper Y chromosome segregation. Notably, HP1D2 is a paralog of rhino, a gene involved in activating the transcription of piRNA clusters, which generate piRNA-class small RNAs in Drosophila [18,22]. In the Winters and Durham systems, recent studies have uncovered the crucial roles of the endogenous RNA-interference (RNAi) pathway in defending X-linked meiotic drive genes from the Distorter on the X (Dox) family drivers [23-25]. Interestingly, evolutionary tracing of the origins of the Dox family genes in D. simulans revealed their relationship to Protamine, a gene involved in sperm chromatin packaging [24,26].

A typical pattern observed in the meiotic drivers of D. melanogaster and D. simulans is that these genes are often truncated or duplicated versions of otherwise normal genes essential for cellular and developmental functions. Thus, the mechanisms of how meiotic drive genes unleash selfish activities to compromise gametogenesis remain unclear. Nevertheless, studies in the melanogaster complex have advanced our understanding of the suppression of sex chromosome meiotic drive. While the primary role of the piRNA pathway is to suppress TE activity in the germline, recent studies in Drosophila have uncovered a sexually dimorphic piRNA program with distinct functions during male gametogenesis, including its involvement in suppressing meiotic conflicts [27,28]. Thus, these studies have extended our understanding of piRNA pathway silencing beyond TEs.

In contrast, the RNAi pathway, where small-interfering RNAs (siRNAs) mediate silencing, has been widely employed as a powerful experimental gene silencing tool for the past two decades, though its endogenous utilities have remained mysterious. Like the piRNA pathway, the RNAi pathway is an ancient small RNA-mediated defense mechanism against genome invaders. However, the substrate for the generation of small RNAs and the Argonaute family proteins that siRNAs and piRNAs associate with are distinct between these two pathways (reviewed in [29]). In D. simulans, the RNAi pathway was shown to be crucial to controlling X-linked meiotic drivers from the Dox family, thereby safeguarding the integrity of male gametogenesis [23-26]. Furthermore, the unsuspected roles of the RNAi pathway in defending intragenomic conflicts may be more widespread than previously thought, as studies in plants have recently demonstrated its crucial role in silencing pollen drive during fertilization [30].

This review aims to highlight the emerging and crucial roles of small RNA-mediated suppression of meiotic conflicts during male gametogenesis, focusing on studies from Drosophila, notably D. melanogaster and closely related simulans clade, which consists of three species, namely, D. simulans, D. mauritania, and D. sechellia (Figure 1A). Selfish genes like meiotic drivers impose a net negative fitness effect on reproductive function. Therefore, the evolution of novel suppression strategies serves to maintain optimal reproductive fitness and inevitably shapes the evolution of germline development and reproduction. As a tangential perspective, the review also aims to shed light on the emerging understanding of how the rapid co-evolutionary dynamics between meiotic drive and suppression affect genome evolution and speciation.

The Ste locus and its suppressor, Su(Ste), are testis-expressed tandem duplications on the X and Y chromosomes, respectively [31]. Ste duplicate copies are related to a testis-specific paralog of β subunit of protein kinase, CkIIβ [32], and the suppression of X-linked Ste by Y-linked Su(Ste) is found only in D. melanogaster, although homologous sequences are found on the X and Y in other simulans-clade species [33,34]. This indicates that the Ste and Su(Ste) silencing via piRNAs is a unique adaptation in D. melanogaster to mitigate sex chromosome conflict. The X- and Y-linked copies emerged from the duplication of suppressor of Ste-like (Ssl)—an autosomal paralog of CkIIβ2 that first duplicated to the Y-chromosome, followed by amplification on the X and Y chromosomes [14] (Figure 1B). Deletions encompassing the Su(Ste) locus on the Y chromosome result in the derepression of X-linked Ste copies, leading to meiotic defects during male gametogenesis [31,35].

Genetic studies of chromosome segregation in Su(Ste) mutant males found that both sex chromosomes and autosomes undergo non-disjunction (i.e. fail to segregate properly during meiosis), revealing a phenotype consistent with meiotic drive [35]. In addition, the derepression of the Ste locus on the X-chromosome leads to needle-shaped crystalline aggregates in the nuclei and cytoplasm of primary spermatocytes. Critically, the severity of the phenotype depended on the strength of allelic states and copy number of the Ste locus [32]. Combined with the male sterility phenotype observed in Su(Ste) mutant males, these observations led to the notion that Ste is a remnant of intragenomic conflicts between the sex chromosomes in D. melanogaster [12,13].

Interestingly, the suppressor, Su(Ste) on the Y-chromosome lacks protein-coding potential and is highly divergent at the sequence level compared with the X-linked Ste copies [36]. Due to the presence of multiple copies on both genomic strands, Su(Ste) produces sense and antisense RNA complementary to Ste, which are processed into small RNAs and mediate the silencing of Ste genes on the X chromosome (Figure 1C) [16,37]. Indeed, pioneering studies on the Stellate system in D. melanogaster led to identifying a germline-specific small RNA pathway—now widely known as the piRNA pathway [16,36,38]. Additional studies have since revealed the essential role of piRNA pathway in suppressing TEs in the germ line, a highly conserved function across animals [39]. piRNA-mediated target silencing occurs through the assembly of an RNA-induced silencing complex (RISC), in which the Argonaute family proteins—Aub or Ago3—bind to piRNAs and silence complementary target RNAs via post-transcriptional silencing (Figure 1C). In contrast, Piwi, another Argonaute member in the piRNA pathway, is involved in co-transcriptional silencing in the nucleus [40].

In addition to understanding sex chromosome meiotic conflicts, the Stellate system in Drosophila has also served as an excellent model to investigate the sex-specific mechanisms of piRNA-mediated post-transcriptional silencing. For instance, in Drosophila, maternally derived piRNAs have an epigenetic role in triggering piRNA production for effective TE silencing in the progeny [41]. If so, how Y-linked piRNA production from the Su(Ste) could be initiated remained an unsolved question. Interestingly, a recent study revealed that Su(Ste) copies on the Y chromosome, which have a 1360/Hoppel TE insertion at the 5′ end adjacent to Su(Ste), act as a trigger. Here, maternal 1360/Hoppel piRNAs guide the production of Su(Ste) piRNAs, leading to Ste silencing in males [42]. These recent studies on the Stellate system highlight the highly adaptable nature of piRNA-mediated silencing in mitigating active and emerging meiotic conflicts.

While extensive research has uncovered the intricacies of the piRNA pathway’s role in TE silencing, the emerging mechanics of relaxed piRNA-targeting rules [43] suggests that piRNA-mediated silencing is a highly adaptable system capable of targeting rapidly diverging sequences—an evolutionary hallmark of genes like Ste involved in intragenomic conflicts. However, how often the piRNA pathway gets recruited to silence endogenous selfish genes like Ste and, in general, sex chromosome conflicts remains unclear. New studies examining the sex-specific piRNA program in the Drosophila male germline have uncovered piRNA targeting of novel endogenous genes such as nod and paics, and petrel, which targets the endogenous gene pirate, appears to be multi-copy genes on the sex chromosomes like Ste [27]. However, it remains to be determined whether these novel endogenous piRNA targets play a role in meiotic conflicts. Nevertheless, the role of piRNA pathway in defending endogenous selfish genes has expanded our understanding of piRNA targeting beyond TEs, highlighting its crucial function in resolving intragenomic conflicts between the sex chromosomes.

The hybrid male sterility resulting from inter-species hybridization among the three species in the simulans clade (Figure 1A) makes it an excellent model for studying the genetics of speciation [44]. These studies led to the original observations of sex-ratio distortion and the discovery of sex chromosome drive in D. simulans. In the Durham drive system, a dominant suppressor called Too Much Yin (Tmy) was found to inhibit X-linked drive, although the specific meiotic driver(s) on the X chromosome has not been identified [19]. Similarly, for the Winters system, an inverted repeat suppressor, Not Much Yang (Nmy) was found to suppress an X-linked driver called Dox, suggesting the likely involvement of homology-dependent silencing in controlling sex chromosome drive [20,21]. Homology analyses could not identify the orthologs of Dox in D. melanogaster, suggesting that Dox might be a novel gene in D. simulans. Interestingly, the discovery of a paralog of Dox, known as Mother of Dox (MDox), led to the notion that the meiotic drive gene Dox evolved from its ancestral gene, MDox [21]. Further investigations clarified that the Nmy locus encodes hairpin RNA (hpRNA) class endo-siRNAs [45], which are processed into predominantly 21 nucleotide siRNAs by the RNAi machinery that targets Dox and MDox in trans for silencing (Figure 2A) [23]. The cellular RNAi toolkit encompasses the ribonuclease Dcr2, which generates small RNAs from double-stranded RNA substrates that are then loaded onto the Argonaute family member Ago2 for RISC activity (Figure 2B) [46]. Interestingly, in nmy mutants, both Dox and MDox are derepressed [23], resulting in a sex-ratio distortion phenotype. This suggested that MDox may also function as a meiotic driver, although the individual contributions of Dox and MDox to the drive phenotype are yet to be determined. Furthermore, genomic analyses revealed that the Tmy locus encodes a novel hpRNA and displays significant homology to Nmy, suggesting that the Winters and Durham drive systems are evolutionarily inter-connected. Indeed, improved genome assemblies in D. simulans [47] identified two additional genes related to Dox and MDox, namely, ParaDox (PDox1 and PDox2), on the X-chromosome that are highly homologous to Tmy, suggesting that the different Dox family genes and their concomitant hpRNA suppressors may have originated during repeated bouts of meiotic drive and suppression (Figure 2A) [24,26].

Since the closest sister species, D. melanogaster, lacks any of these hpRNA suppressors, the origins of the Dox family genes remained unclear for over a decade. Detailed evolutionary tracing led to the identification that the meiotic driver Dox emerged via a chimeric fusion by acquiring segments from multiple D. melanogaster genes with its open-reading frame originating from Protamine [24,26]. Protamines are sperm nuclear basic proteins (SNBPs) involved in chromatin compaction. Many SNBP factors are essential for male fertility and are known to exhibit signatures of rapid evolution [48,49]. Dox family genes encode a high mobility group (HMG)-box domain that is predicted to bind DNA—a domain typically found in many transcription factors and, more importantly, in Protamine-like SNBPs [50]. However, the HMG-box domain in Dox appears to be highly divergent from the Protamine, suggesting rapid diversification of Dox family genes, likely due to its role in intragenomic conflicts.

Mechanistically, how Protamine-like Dox family genes derail male gametogenesis is yet to be understood; however, the chromatin association of PDox2 during telophase of meiosis I and II lends support to their potential roles in the misregulation of the chromatin landscape during spermiogenesis [25]. In contrast, Dox sub-cellular localization was found to be primarily cytoplasmic during meiosis and was not detected during postmeiotic stages, while canonical protamines exhibit exclusive nuclear localization during postmeiotic stages [25]. In line with these observations, the loss of tmy and nmy hpRNA has marked differences in spermiogenic phenotypes exhibiting male sterility and sex-ratio distortion, respectively, due to derepression of distinct Dox family genes (Figure 2C). These findings suggest that although Dox factors have originated from protamines, their subcellular localization paints a complex picture consistent with likely neo-functionalization. Therefore, whether the Dox family members may function as protamine mimics or have acquired other novel function(s) is yet to be determined.

Furthermore, comparative evolutionary analyses of Dox family genes have revealed that the drive and suppression system originated in the simulans-clade ancestor [24]. Interestingly, the Dox family genes have undergone massive amplification on the X chromosomes in D. mauritiana and D. sechellia, with 22 additional copies identified across both genomes. In most instances of these amplified copies, the Dox family duplicates lack synteny and orthology, indicating that multiple independent insertions have occurred across all three simulans-clade species. Detailed evolutionary tracing on the origins of Dox family genes has also revealed the association of Dox genes with a family of satellite repeats called 359-satellite, which led to the amplification of these genes on the X-chromosome [51]. Crucially, the expanding cohort of Dox genes has also birthed novel Tmy-like hpRNAs, namely, Tmy2 in D. mauritiana and m-Tmy-C in D. sechellia.

The rapid expansion of a family of meiotic drivers and their hpRNA suppressors is a testament to an ongoing genetic arms race in the simulans clade. Indeed, CRISPR-Cas9 mutagenesis of the core RNAi factor Dcr2 in D. simulans uncovered many de novo hpRNAs like Nmy and Tmy with predominantly X-linked targets. These novel hpRNAs and their targets are absent in D. melanogaster, indicating RNAi pathway’s unexpected broader role in mitigating other likely meiotic conflicts beyond the Dox family genes in D. simulans [52]. Thus, compromising the RNAi machinery in D. simulans unveiled novel, dynamically evolving hpRNA-target networks, highlighting how germline genetic conflicts can invariably alter genome evolution among closely related and incipient Drosophila species.

Past and recent research on sex chromosome drive in Drosophila has uncovered common themes, particularly from the detailed study of a few examples in D. melanogaster and D. simulans. First, genes identified to cause sex chromosome drive in these species tend to be duplicates of ordinary genes with a history of amplification on the sex chromosomes [24,26,36]. Interestingly, another well-known example of an autosomal meiotic driver in the segregation distortion (SD) system in D. melanogaster is a Ran GTPase activating protein (RanGAP) [53,54]. RanGAP is also seemingly a normal gene, which acts as a GTPase activator for the nuclear Ras-related regulatory protein Ran [55]. Interestingly, SD-mediated meiotic drive results in the compromised histone-to-protamine transition [56], and knockdown of protamines in D. melanogaster was shown to induce distortion in the SD background [57]. These studies highlight a recurring theme in connecting protamines to meiotic drive. On the connected theme of protamines, Y-linked protamine-like Mst77Y, which are paralogs of autosomal Mst77F required for histone-to-protamine transition, were shown to compromise the compaction of the X-chromosome when misexpressed [58]. Although the effect was modest on sex-ratio distortion, the regulation of Mst77Y is mediated by a nucleolar protein called Modulo, highlighting other potential means to resolve meiotic conflicts that do not involve small RNA pathways.

The second common trend observed is the association of meiotic drive genes with repetitive DNA, either as a template for amplification, as in the case of the Dox system, or for regulation, as exemplified by the 1360/Hoppel repeats in Su(Ste) system. Interestingly, the strength of meiotic drive in the SD system is also associated with Responder (Rsp) satellite repeats, highlighting genomic repeats as significant players in the emergence and maintenance of meiotic drive. Therefore, one of the future challenges is to uncover how (and when) duplicate or ampliconic genes acquire selfish properties and what roles genomic repeats may play in the evolution of meiotic drive.

Studies in D. melanogaster and D. simulans have also uncovered the crucial importance of small RNA pathways in defending against meiotic drive. Both piRNA and siRNA pathways are deeply conserved in evolution [39], and the recruitment of piRNA pathway to silence Stellate genes in D. melanogaster and the RNAi machinery in counteracting Dox-mediated sex chromosome drive in D. simulans attest to the evolutionary plasticity in the use of different small RNA pathways for silencing intragenomic conflicts. These studies also highlight how small RNA-based silencing strategies can be a quick and potent response against emerging meiotic conflicts and may explain their deep conservation across evolution and sexually dimorphic functions, as revealed by a recent study [59].

Indeed, the remarkable flexibility in recruiting different small RNA pathways to combat potential meiotic conflicts was recently shown for the endogenous gene, pirate, in D. melanogaster, targeted by Y-linked piRNAs in the male germline. Interestingly, in the closest sister species D. mauritiana, pirate is targeted by siRNAs instead of piRNAs, highlighting the variability in the use of different small RNA pathways to target the same gene [27]. Thus, in the evolution’s toolkit to defend against emerging meiotic conflicts, these small RNA pathways can be viewed as a ‘Swiss Army Knife’ for effectively responding to intragenomic conflicts. Therefore, the observations from Drosophila studies raise the question of how commonly small RNA pathways combat sex chromosome conflicts and meiotic drive during male gametogenesis. With high-quality genome assemblies becoming available in many non-model Drosophila species [60], small RNA profiling from the male germline in multiple Drosophila species and other dipteran insects may help uncover the utility and variation among these pathways in targeting endogenous selfish genes.

Finally, studies on sex chromosome drive in Drosophila have uncovered surprisingly rapid evolutionary dynamics between selfish genes and their small RNA suppressors. Thus, it is becoming increasingly apparent that these male meiotic conflicts can potently shape the evolutionary trajectories of genes and genomes. For instance, the Dox family meiotic drive genes and their concomitant suppressors in D. simulans are not only lineage-specific but also exhibit remarkable variation in terms of expansion in copy numbers of Dox factors and the evolution of multiple Tmy-like suppressors in the simulans clade that emerged from repeated cycles of meiotic drive and suppression [24,26].

One of the consequences of rapid evolution from intragenomic conflicts is that the drive and suppression can compromise both reproductive fitness in the species harboring these drive elements and during hybridization between closely related species. Indeed, in a few examples from non-model Drosophila species, sex chromosome drive is either directly associated with hybrid sterility [61] or the sex chromosome meiotic driver is causal for reproductive isolation [62,63]. However, how frequently meiotic drive acts as an evolutionary force to propel reproductive isolation and speciation remains to be investigated. As sex chromosome drive is known to occur in many Drosophila species and dipteran insects [64,65], future studies on small RNA profiling, in conjunction with high-quality genome assemblies from these species, could answer the breadth of the usage of RNAi or piRNA pathways in mitigating meiotic drive. Recently, the expanding scope of genome sequencing and small RNA profiling from insects such as Bombyx mori and Plutella xylostella have linked a role for piRNAs in reproduction and the sex determination cascade [66,67]; however, it remains to be seen how frequently small RNA pathways are involved in suppressing meiotic sex chromosome conflicts.

The authors declare that there are no competing interests associated with the manuscript.

Thanks to Peiwei Chen, Cécile Courret, and two anonymous reviewers for their comments and feedback on the manuscript. J.V. acknowledges financial support from the National Institutes of Health (GM137007), the University of Texas Rising STARs program, and the University of Texas at San Antonio. The author apologizes to colleagues whose contributions to this field were not cited in this review article due to space constraints.

Dox

Distorter on the X

HMG

high mobility group

MDox

Mother of Dox

Nmy

Not Much Yang

RNAi

RNA-interference

RanGAP

Ran GTPase activating protein

Rsp

Responder

SD

segregation distortion

SNBPs

sperm nuclear basic proteins

Ste

Stellate

Su(Ste)

Suppressor of Stellate

TEs

transposable elements

Tmy

Too Much Yin

hpRNA

hairpin RNA

piRNA

piwi-interacting RNA

siRNAs

small-interfering RNAs