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Although thousands of long non-coding RNAs (lncRNA) have been identified in the genomes of higher eukaryotes, the precise function of most of them is still unclear. Here, we show that a >65 kb, male-specific, lncRNA, called male-specific abdominal (msa) is required for the development of the secondary cells of the Drosophila male accessory gland (AG). msa is transcribed from within the Drosophila bithorax complex and shares much of its sequence with another lncRNA, the iab-8 lncRNA, which is involved in the development of the central nervous system (CNS). Both lncRNAs perform much of their functions via a shared miRNA embedded within their sequences. Loss of msa, or of the miRNA it contains, causes defects in secondary cell morphology and reduces male fertility. Although both lncRNAs express the same miRNA, the phenotype in the secondary cells and the CNS seem to reflect misregulation of different targets in the two tissues., Author summary In many animals, the male seminal fluid induces physiology changes in the mated female that increase a male’s reproductive success. These changes are often referred to as the post-mating response (PMR). In Drosophila, the seminal fluid proteins responsible for generating the PMR are made in a specialized gland, analogous to the mammalian seminal vesicle and prostate, called the accessory gland (AG). In this work, we show that a male-specific, long, non-coding RNA (lncRNA), called msa, plays a critical role in the development and function of this gland, primarily through a microRNA (miRNA) encoded within its sequence. This same miRNA had previously been shown to be expressed in the central nervous system (CNS) via an alternative promoter, where its ability to repress homeotic genes is required for both male and female fertility. Here, we present evidence that the targets of this miRNA in the AG are likely different from those found in the CNS. Thus, the same miRNA seems to have been selected to affect Drosophila fertility through two different mechanisms. Although many non-coding RNAs have now been identified, very few can be shown to have function. Our work highlights a lncRNA that has multiple biological functions, affecting cellular morphology and fertility.

Endoplasmic reticulum (ER) stress occurs when misfolded proteins accumulate in the ER. The cellular response to ER stress involves complex transcriptional and translational changes, important to the survival of the cell. ER stress is a primary cause and a modifier of many human diseases. A first step to understanding how the ER stress response impacts human disease is to determine how the transcriptional response to ER stress varies among individuals. The genetic diversity of the eight mouse Collaborative Cross (CC) founder strains allowed us to determine how genetic variation impacts the ER stress transcriptional response. We used tunicamycin, a drug commonly used to induce ER stress, to elicit an ER stress response in mouse embryonic fibroblasts (MEFs) derived from the CC founder strains and measured their transcriptional responses. We identified hundreds of genes that differed in response to ER stress across these genetically diverse strains. Strikingly, inflammatory response genes differed most between strains; major canonical ER stress response genes showed relatively invariant responses across strains. To uncover the genetic architecture underlying these strain differences in ER stress response, we measured the transcriptional response to ER stress in MEFs derived from a subset of F1 crosses between the CC founder strains. We found a unique layer of regulatory variation that is only detectable under ER stress conditions. Over 80% of the regulatory variation under ER stress derives from cis-regulatory differences. This is the first study to characterize the genetic variation in ER stress transcriptional response in the laboratory mouse. Our findings indicate that the ER stress transcriptional response is highly variable among strains and arises from genetic variation in individual downstream response genes, rather than major signaling transcription factors. These results have important implications for understanding how genetic variation impacts the ER stress response, an important component of many human diseases., Author Summary Genetic variation among individuals can greatly impact the severity of disease outcomes. To understand the effects of different genetic backgrounds on disease presentation, we focused on ER stress, an important cellular stressor that impacts many human diseases. We examined how genetic variation affects ER stress response, at the RNA level, in eight laboratory mouse strains and their hybrid progeny. We find that each mouse strain responds in a unique way to ER stress, and we characterized the patterns of genetic variation that underlie the differences in ER stress response between the strains. We find that the strains show major differences in their inflammatory response to ER stress, a critical component to disease. The results of this study are important for understanding potential ways in which genetic variation in ER stress response could impact disease, and lays the groundwork for future studies in human patients.

Seminal fluid proteins transferred from males to females during copulation are required for full fertility and can exert dramatic effects on female physiology and behavior. In Drosophila melanogaster, the seminal protein sex peptide (SP) affects mated females by increasing egg production and decreasing receptivity to courtship. These behavioral changes persist for several days because SP binds to sperm that are stored in the female. SP is then gradually released, allowing it to interact with its female-expressed receptor. The binding of SP to sperm requires five additional seminal proteins, which act together in a network. Hundreds of uncharacterized male and female proteins have been identified in this species, but individually screening each protein for network function would present a logistical challenge. To prioritize the screening of these proteins for involvement in the SP network, we used a comparative genomic method to identify candidate proteins whose evolutionary rates across the Drosophila phylogeny co-vary with those of the SP network proteins. Subsequent functional testing of 18 co-varying candidates by RNA interference identified three male seminal proteins and three female reproductive tract proteins that are each required for the long-term persistence of SP responses in females. Molecular genetic analysis showed the three new male proteins are required for the transfer of other network proteins to females and for SP to become bound to sperm that are stored in mated females. The three female proteins, in contrast, act downstream of SP binding and sperm storage. These findings expand the number of seminal proteins required for SP's actions in the female and show that multiple female proteins are necessary for the SP response. Furthermore, our functional analyses demonstrate that evolutionary rate covariation is a valuable predictive tool for identifying candidate members of interacting protein networks., Author Summary Reproduction requires more than a sperm and an egg. In animals with internal fertilization, other proteins in the seminal fluid and the female are essential for full fertility. Although hundreds of such reproductive proteins are known, our ability to understand how they interact remains limited. In this study, we investigated whether shared patterns of protein sequence evolution were predictive of functional interactions by focusing on a small network of proteins that control fertility and female post-mating behavior in the fruit fly, Drosophila melanogaster. We first showed that the six proteins already known to act in this network display correlated patterns of evolution across the Drosophila phylogeny. We then screened hundreds of otherwise uncharacterized male and female reproductive proteins and identified those with patterns of evolution most similar to those of the known network proteins. We tested each of these candidate genes and found six new network members that are each required for long-term fertility. Using molecular genetics, we also observed that the steps in the network at which these new proteins act are consistent with their strongest evolutionary correlations. Our results suggest that patterns of coevolution may be broadly useful for predicting protein interactions in a variety of biological processes.

Proteases and protease inhibitors have been identified in the ejaculates of animal taxa ranging from invertebrates to mammals and form a major protein class among Drosophila melanogaster seminal fluid proteins (SFPs). Other than a single protease cascade in mammals that regulates seminal clot liquefaction, no proteolytic cascades (i.e. pathways with at least two proteases acting in sequence) have been identified in seminal fluids. In Drosophila, SFPs are transferred to females during mating and, together with sperm, are necessary for the many post-mating responses elicited in females. Though several SFPs are proteolytically cleaved either during or after mating, virtually nothing is known about the proteases involved in these cleavage events or the physiological consequences of proteolytic activity in the seminal fluid on the female. Here, we present evidence that a protease cascade acts in the seminal fluid of Drosophila during and after mating. Using RNAi to knock down expression of the SFP CG10586, a predicted serine protease, we show that it acts upstream of the SFP CG11864, a predicted astacin protease, to process SFPs involved in ovulation and sperm entry into storage. We also show that knockdown of CG10586 leads to lower levels of egg laying, higher rates of sexual receptivity to subsequent males, and abnormal sperm usage patterns, processes that are independent of CG11864. The long-term phenotypes of females mated to CG10586 knockdown males are similar to those of females that fail to store sex peptide, an important elicitor of long-term post-mating responses, and indicate a role for CG10586 in regulating sex peptide. These results point to an important role for proteolysis among insect SFPs and suggest that protease cascades may be a mechanism for precise temporal regulation of multiple post-mating responses in females., Author Summary Proteases can destroy, activate, or otherwise modulate the function of other proteins. In seminal fluid, many proteins have to be activated or degraded after mating; proteolysis is an effective way to accomplish this because seminal fluid proteins act outside of the cell, where most other regulatory processes cannot be used. Despite the presence of proteases in the seminal fluid of many animals, nearly nothing is known about the kinds of processes they regulate. Here, we present evidence of a protease cascade in the seminal fluid of the fruit fly Drosophila melanogaster. This cascade involves two proteases that are activated during mating. Once in the female, the downstream protease acts on two other proteins that are important for ovulation and sperm storage. Interestingly, the protease at the top of the cascade, CG10586, is also required for other female post-mating responses, including egg laying and sperm usage, independent of the second protease. Thus, CG10586 might be a general regulatory switch used by the male to quickly activate many female responses after mating.

Successful reproduction is critical to pass genes to the next generation. Seminal proteins contribute to important reproductive processes that lead to fertilization in species ranging from insects to mammals. In Drosophila, the male's accessory gland is a source of seminal fluid proteins that affect the reproductive output of males and females by altering female post-mating behavior and physiology. Protein classes found in the seminal fluid of Drosophila are similar to those of other organisms, including mammals. By using RNA interference (RNAi) to knock down levels of individual accessory gland proteins (Acps), we investigated the role of 25 Acps in mediating three post-mating female responses: egg production, receptivity to remating and storage of sperm. We detected roles for five Acps in these post-mating responses. CG33943 is required for full stimulation of egg production on the first day after mating. Four other Acps (CG1652, CG1656, CG17575, and CG9997) appear to modulate the long-term response, which is the maintenance of post-mating behavior and physiological changes. The long-term post-mating response requires presence of sperm in storage and, until now, had been known to require only a single Acp. Here, we discovered several novel Acps together are required which together are required for sustained egg production, reduction in receptivity to remating of the mated female and for promotion of stored sperm release from the seminal receptacle. Our results also show that members of conserved protein classes found in seminal plasma from insects to mammals are essential for important reproductive processes., Author Summary In sexually reproducing organisms, sperm enter the female in combination with seminal proteins that are critical for fertility. These proteins can activate sperm or enhance sperm storage within the female, and can improve the chance that sperm will fertilize eggs. Understanding the action of seminal proteins has potential utility in insect pest control and in the diagnosis of certain human infertilities. However, the precise function of very few seminal proteins is known. To address this, we knocked down the levels of 25 seminal proteins individually in male fruit flies, and tested the males' abilities to modulate egg production, sperm storage/release, or behavior of their mates. We found five seminal proteins that are necessary to elevate offspring production in mated females. Four of these proteins are needed for efficient release of sperm from storage to fertilize eggs, a function that had not been previously assigned to any seminal protein. All four are in biochemical classes that are conserved in seminal fluid from insects to humans, suggesting they may play similar sperm-related roles in other animals. In addition to assigning functions to particular seminal proteins, our results suggest that fruit flies can serve as a model with which to dissect the functions of conserved protein classes in seminal fluid.