BIOLOGICAL OVERVIEW

A hallmark of germline cells across the animal kingdom is the presence of perinuclear, electron-dense granules called nuage. In many species examined, Vasa, a DEAD-box RNA helicase, is found in these morphologically distinct particles. Despite its evolutionary conservation, the function of nuage remains obscure. A null allele of maelstrom (mael) has been characterized. Maelstrom protein is localized to nuage in a Vasa-dependent manner. By phenotypic characterization, maelstrom has been defined as a spindle-class gene that affects Vasa modification. In a nuclear transport assay, it has been determined that Maelstrom shuttles between the nucleus and cytoplasm, which may indicate a nuclear origin for nuage components. Interestingly, Maelstrom, but not Vasa, depends on two genes involved in RNAi phenomena for its nuage localization: aubergine and spindle-E (spn-E). Furthermore, maelstrom mutant ovaries show mislocalization of two proteins involved in the microRNA and/or RNAi pathways, Dicer and Argonaute2 (see Argonaute I), suggesting a potential connection between nuage and the microRNA-pathway (Findley, 2003).

How germline status is established and maintained in sexually reproducing organisms is a fundamental question in developmental biology. A conserved feature of germ cells in species across the animal kingdom is the presence of a distinct morphological element called nuage. Ultrastructurally, nuage appears as electron-dense granules that are localized to the cytoplasmic face of the nuclear envelope. Despite the breadth of nuage in the animal kingdom, there is currently a lack of depth in understanding its function. In animals ranging from the nematode to vertebrates, the Vasa protein has been detected in these granules. Both nuage and Vasa thus offer potential clues as to what makes a germ cell unique (Findley, 2003).

One system with high potential for understanding the role of nuage is Drosophila. In females, Vasa-positive germline granules are continuously present throughout the life cycle, taking one of two forms, nuage or pole plasm. Pole plasm, which contains polar granules, is a determinant that is both necessary and sufficient to induce formation of the germ lineage in early embryogenesis. In Drosophila, nuage is first detectable when primordial germ cells are formed; it persists through adulthood, where it is present in all germ cell types of the ovary (Findley, 2003).

In Drosophila, three proteins are known to localize to nuage: Vasa, Aubergine and Tudor. The sequence or mutant phenotype of each gene suggests a role in post-transcriptional RNA function. Vasa is a DEAD-box RNA helicase required for nurse cell-to-oocyte transport of several mRNAs critical to oocyte patterning. Vasa is also required for efficient translation of several key proteins in oogenesis, and itself interacts both physically and genetically with a Drosophila homolog of yeast, Translation Initiation Factor 2 (dIF2). Vasa is thus potentially implicated in translational control. Aubergine is a member of the RDE1 (for RNAi defective)/AGO1 (Argonaute1) protein family, homologs of which are required in both RNAi and developmental processes in diverse organisms. Aubergine is required, during oogenesis, for efficient translation of Oskar, which is pivotal in initiating pole plasm assembly. Aubergine is also required for RNAi in late oogenesis. Tudor, a novel protein, comprises ten copies of an ~120 residue motif (the 'Tudor Domain', pfam00567) present in several proteins involved or implicated in RNA-binding capacities. The domain has been suggested to mediate protein-protein interactions. Drosophila Tudor is required to mediate transfer of mitochondrial ribosomal RNAs from mitochondria to the surface of polar granules during pole cell formation in early embryogenesis (Amikura, 2001). A role for Tudor prior to pole plasm assembly, however, has not been described (Findley, 2003 and references therein).

Aubergine and vasa are members of a larger group of female sterile mutants, the spindle (spn) class, which produces eggshells with variable anteroposterior (AP) and dorsoventral (DV) axis defects. In most of the characterized spn mutants, the etiology of these patterning defects has be traced to a failure in Gurken (a TGFalpha homolog) presentation in developing egg chambers. Each spn mutant also has a meiotic progression defect: by stage 4 in a normal egg chamber, the DNA within the oocyte nucleus (germinal vesicle) condenses into a compact sphere called a karyosome after successful meiotic recombination. Mutants in all spn genes fail to form a karyosome. The characterized spn genes fall into two groups: those whose gene products are directly required for recombinational DNA repair steps within the germinal vesicle [e.g. okra (okr), spn-B and spn-C] and those whose protein sequence or localization suggest indirect involvement in meiotic progression [e.g. aubergine, vasa and spn-E]. The spn mutants, as a group, demonstrate that meiotic and patterning processes intersect during oogenesis (Findley, 2003).

A null allele of the maelstrom gene, which encodes a novel protein with a human homolog, has been identified and characterized. The mutant displays each of the defects in oocyte development common to the spindle-class. Maelstrom localizes to nuage in a Vasa-dependent manner and maelstrom is required for proper modification of Vasa. Through mutant analysis, this study begins to unravel genetic dependencies of nuage particle assembly (Findley, 2003).

It is unknown whether the known nuage proteins act in a common pathway before their convergence in nuage particles. To begin to answer this question, attempts were made to determine genetic dependencies for nuage particle assembly. AubGFP is known to depend on vasa function for its nuage localization (Harris, 2001). Maelstrom and Vasa localization were analyzed in wild-type, maelstrom, vasa, aubergine and spn-E backgrounds. Maelstrom protein levels proved to be quite variable among ovarioles of single mutant backgrounds. So, in order to compare localization, individual ovarioles were examined in which Maelstrom levels were not significantly reduced. Virtually no Maelstrom immunoreactive signal is present in maelstrom mutants, whereas Vasa is largely maintained in nuage. By contrast, the perinuclear accumulation of Maelstrom is virtually absent in the vasa null mutant, suggesting that Maelstrom localization in nuage is Vasa dependent. Maelstrom's distribution was examined in several vasa point mutants, in the hope of correlating functional domains in the protein with nuage organizational function. Of particular interest were two vasa EMS alleles, vas⁰¹¹ and vas⁰¹⁴, each of which produces a protein devoid of RNA binding and unwinding activities. In both of these mutants, Vasa and Maelstrom colocalization in nuage is largely maintained. Vasa and Maelstrom localization were analyzed in several allelic combinations of aubergine, since a null for this gene has not been described. Both aub^HN2 and aub^N11 alleles encode truncated proteins. In this and other aubergine mutant combinations, the normal concentration of Maelstrom in nuage is severely depleted in all germline cells. Vasa is largely maintained in perinuclear localization in this mutant background, but the normally discrete particles are less obvious; instead, Vasa appears as a more uniform perinuclear band (Findley, 2003).

Spn-E encodes a putative Dex/hD-box RNA helicase, required for proper localization of several oocyte-destined RNAs and proteins over the course of oogenesis. While the localization of Spindle-E in the ovary has not been determined, its involvement in both RNAi and oogenesis, like Aubergine, prompted its inclusion in this analysis. As with aubergine mutants, the concentration of Maelstrom in perinuclear particles is lost in strong spn-E allelic combinations, spn-E^{616/hlsDelta125} and spn-E^{hls3987/hlsDelta125}. Vasa retains a perinuclear concentration in spn-E ovaries, but as in aubergine, the normal particulate appearance of nuage is less pronounced. Localization analysis has been extended to include the remaining members of the better characterized spn-class mutants, spn-A, spn-B, spn-C, spn-D and okr. Of particular interest was spn-B, which has been shown to modify Vasa as a consequence of meiotic checkpoint activation. The dependency of Maelstrom on Vasa for its localization could, in principle, be affected if Vasa is aberrant. However, in multiple allelic combinations of well-characterized spn genes (spn-B, spn-D and okr) and uncloned spn genes (spn-A and spn-C), colocalization of Vasa and Maelstrom in nuage particles was unperturbed at all stages of oogenesis (Findley, 2003).

Normally, a variety of RNAs required for proper oocyte function are transcribed, exported from nurse cell nuclei and transported to the oocyte. Since this process is defective in vasa ovaries, transport in maelstrom ovarioles was assessed, with the aim of resolving potential functions of individual nuage components. It was found that transport of several of these mRNAs, including oskar, orb and BicD, is unaltered in maelstrom ovaries (Findley, 2003).

The dissociation of Maelstrom from nuage particles in aubergine and spn-E backgrounds was intriguing in light of their requirement in RNAi in Drosophila spermatogenesis and late oogenesis. Importantly, proteins (or homologs) of RNAi pathway components also act in micro RNA (miRNA) processing. Since miRNAs have been shown to regulate RNA translation, it is conceivable that miRNAs are assembled in RNP particles formed in nuage. In this setting, nuage could represent a step in the generation of specificity in translational control in the germline. To explore this potential relationship between nuage and RNAi/miRNA processing pathways, the localization of additional RNAi components was examined in wild-type and maelstrom ovaries. Argonaute1 and Argonaute2 are RDE1/AGO1 homologs required for RNAi in Drosophila. Dicer is the core RNase of RNAi in Drosophila; it is also required for production of the small RNA effectors of the RNAi and miRNA pathways in C. elegans. In vertebrate cell lines, Dicer is primarily cytoplasmic. In wild-type Drosophila ovarioles, Dicer and AGO1 appear uniform and cytoplasmic in nurse cell cytoplasm; AGO2 appears cytoplasmic but relatively more granular. In maelstrom ovaries, AGO1 distribution is relatively unperturbed. However, AGO2 and Dicer are both dramatically mislocalized in maelstrom ovarioles. Beginning around stage 3, Dicer aggregates in discrete, often perinuclear foci in nurse cells. AGO2 is observed in perinuclear regions of nurse cells, which, by contrast, can colocalize with Vasa in nuage (Findley, 2003).

The failure of maelstrom oocytes to proceed to the karyosome stage, to establish cytoplasmic polarity and to accumulate Gurken qualifies the inclusion of maelstrom in the spindle class. Maelstrom is a component of Drosophila nuage and is required for proper modification (or processing) of a key nuage component, Vasa. Maelstrom is also present within the nucleus and cytoplasm of all germline cells, and can shuttle between these compartments in a CRM1-dependent manner. Of the known nuage-localizing proteins, Vasa appears to be a pivotal organizer or nucleator of nuage, whereas Maelstrom can be dissociated from nuage particles in aubergine and spn-E mutants. Furthermore, Dicer and AGO2 are mislocalized in the maelstrom background (Findley, 2003).

The characterized spn genes currently fall into two general classes: those that encode proteins that are likely to be directly involved in meiotic recombinational repair, such as okr, spn-B and spn-C; and those, such as maelstrom and vasa, whose mutant meiotic phenotype, protein sequence and/or localization suggest indirect roles. Work presented in this study suggests that the spn mutants can be sorted by an additional criterion: those that are also required for nuage assembly (vasa, aubergine, maelstrom and spn-E) and those that are not (spn-A, spn-B, spn-C, spn-D and okra). Taken together, these data suggest that the Vasa-like group of spn genes are essential in general 'nuage activities' in all cells of the germline. The activity of the spn-B-class genes, which are involved in recombination or meiotic checkpoint, could represent one avenue through which to use or modulate existing nuage functions that are operative within the germline cyst as a whole. Such nuage-related processes, if inactivated or defective, might culminate in polarity and translational defects within the oocyte (Findley, 2003).

Within the time frame of the life cycle of the female fly, pole plasm, in its mature form, is ephemeral. Nuage, by contrast, is present in morphologically stable form from mid-embryogenesis through to late oogenesis in the adult. Nuage is thus a feature that is specific to established germ cells, perhaps reinstated by pole plasm components in early embryogenesis. Maelstrom is unique among previously identified Drosophila nuage components, which are also concentrated in pole plasm. Maelstrom is thus the first identified nuage-specific component in Drosophila. Nuage is the most conserved form of the Vasa-positive germline granule: it is present in germ cells of diverse organisms, including mammals, which do not use a pole plasm equivalent. The cell biology of nuage, however, remains largely unexplored. Since nuage components are also present in the nucleus and cytoplasm, these particles may represent a morphologically distinct form (or kinetic intermediate) of a nucleocytoplasmic continuum. Maelstrom can shuttle between nuclear and cytoplasmic compartments. The observation that Vasa shows only a slight redistribution to the nuclear compartment implies that these proteins are not necessarily always associated. This begs the question of whether components of nuage converge on and diverge from perinuclear particles from separate pathways or subcellular compartments. As more nuclear transport tools become available, how these particles are formed will be better resolved (Findley, 2003).

Within egg chambers, a number of mRNAs crucial for oocyte patterning are synthesized in nurse cells and transported to the developing oocyte. The oocyte represents a discrete compartment in the continuous cytoplasm of the germline cyst, and precise spatiotemporal control of nurse cell-derived mRNA translation is crucial for proper development. Between transcription in nurse cell nuclei and their ultimate translation, oocyte-destined RNAs are likely to be associated with factors required to mediate both localization and translational control. Heterogeneous nuclear ribonucleoproteins (hnRNP-proteins) associated with mRNAs during their transit from nucleus to cytoplasm can play key roles in mRNA localization and translational control. Similarly, splicing factors have been shown to continue their association with mRNA into cytoplasm. And although the exchange of nuclear for cytoplasmic RNA-binding proteins has been demonstrated, the location and regulation of such exchange processes are poorly understood. A working hypothesis is that nuage in the Drosophila germline (and other systems) functions in such an exchange process. In this capacity, nuage might be a platform for, or represent a kinetic intermediate of, cytoplasmic ribonucleoprotein (RNP) particle assembly. The perinuclear localization of nuage and the observation that Maelstrom shuttles between compartments are consistent with this role. Two additional lines of evidence support the proposition. The first is that nuage ultrastructurally interfaces with sponge bodies, which are highly abundant, RNA-rich particles present in the cytoplasm of nurse cells and the oocyte. Mounting evidence points to a role for the sponge body as a vehicle for transport of RNP complexes between nurse cells and the oocyte. Sponge bodies are also highly enriched for several regulatory proteins, including Exuperantia, DEAD-box RNA helicase Me31B and cold shock protein YPS, that can be purified as an RNase-sensitive RNP complex. This complex, by purification or localization, is associated with numerous oocyte-destined RNAs, such as oskar and bicoid. Ultrastructurally, the perinuclear fraction of sponge body particles appear to embed individual nuage particles, a morphological association that implicates the interface as one possible segment in what may be a continuous RNP assembly process(Findley, 2003 and references therein).

The second line of evidence is that three of the proteins present in, or required for, nuage particle assembly (Aubergine, Vasa and Spn-E) are also implicated in post-transcriptional RNA-related capacities. Aubergine mutants fail to translate Oskar efficiently, despite proper localization of oskar mRNA to the oocyte posterior. Aubergine is concentrated in two distinct regions within germline cysts: in nuage and also in pole plasm. Thus, aubergine function may be required for proper Oskar translation before oskar mRNA is even transported to the oocyte posterior. Vasa is a DEAD-box RNA helicase, a class of proteins thought to act as RNA chaperones (Findley, 2003 and references therein).

Vasa-null egg chambers display a systemic failure in mRNA targeting. This could be attributable, in part, to the loss of interface of nuage with sponge bodies; vasa null ovaries are devoid of nuage at the ultrastructural level. spn-E encodes a Dex/hD class (putative) RNA helicase. spn-E ovaries contain enlarged sponge bodies and display defects in the localization of several normally oocyte-destined molecules, including bicoid RNA, as well as BicD(GFP) and Dynein Heavy Chain. Each of these molecules is largely retained in mutant nurse cells, accumulating in the vicinity of ring canals. The specificity of the localization defect suggests an underlying defect in transport through ring canals, which is a discrete, microtubule-independent step in nurse cell-to-oocyte transport. This defect (together with the nuage assembly defect of spn-E) is consistent with the hypothesis that nuage may function in formation of RNPs required for correct mRNA localization in the Drosophila germline (Findley, 2003).

A tantalizing possibility for nuage is that it may function at some point in miRNA and/or RNP particle assembly processes. This hypothesis is supported by the mislocalization of Dicer and AGO2 to perinuclear regions of germline cells in the maelstrom mutant. Such discrete redistributions of proteins could reflect an accretion of intermediate in a normally maelstrom-mediated step. A connection is further insinuated by the requirement of both aubergine and spn-E in the assembly of nuage particles. In mutants of both genes, Maelstrom is dissociated from perinuclear Vasa particles in all germline cells. The dissociation is interesting because both aubergine and spn-E have a common requirement in double-stranded RNA-mediated gene silencing in both Drosophila spermatogenesis and late oogenesis. In the testis, spn-E is required for silencing of retrotransposons (e.g. copia) and both spn-E and aubergine are required for silencing of genomic tandem repeats (e.g. Stellate). Mutants in either gene relieve RNAi-mediated suppression of respective target genes. The fact that each is also required for proper mRNA localization or translation raises the possibility that these proteins could function as common components in the allied miRNA pathway. RNAi and miRNA are mechanistically related: each pathway processes a dsRNA substrate, using a common processing factor, Dicer, to generate the respective small RNA effectors of each pathway. In C. elegans, 24 RDE1/AGO1 homologs have been identified. The studied homologs are required in either RNAi or miRNA processing, but in not both pathways. The Drosophila genome encodes only five RDE1/AGO1 homologs: Piwi, Aubergine, AGO1, AGO2 and AGO3, which may necessitate dual usage in both miRNA and RNAi pathways (Findley, 2003).

Recent reports suggest that hundreds of miRNAs exist in metazoans. These miRNAs are thought to be modulators of target mRNA translation, although additional functions have been hypothesized. Indeed, miRNAs might represent a common means of post-transcriptional regulation of gene expression in both vertebrates and invertebrates. It is known for many cell types, including neurons and oocytes, that translation and localization of mRNA is controlled by RNA-binding proteins. However, the specificity of this process is poorly understood. In some cases, a sequence-specific RNA binding protein is found to be involved; in other cases a combinatorial action of many hnRNPs is proposed. The abundance of miRNAs raises the possibility that these small RNAs could generate the missing specificity: miRNAs bound to target mRNAs are predicted to form a loop structure that could be recognized by multiple RNA-binding proteins, allowing for assembly of a full RNP particle. In the context of oogenesis, miRNAs could provide an added level of control by conferring specificity through nucleation or regulated assembly of translational (and possibly localization) control factors on RNAs. The data presented in this study suggest that nuage function may be involved in the miRNA or RNAi pathways. Future experiments should be aimed at determining the role of nuage components in miRNA precursor maturation or in assembly of mature miRNAs with their target mRNAs (Findley, 2003).

Histone acetyltransferase Enok regulates oocyte polarization by promoting expression of the actin nucleation factor spire

KAT6 histone acetyltransferases (HATs) are highly conserved in eukaryotes and have been shown to play important roles in transcriptional regulation. This study demonstrates that the Drosophila KAT6 Enok acetylates histone H3 Lys 23 (H3K23) in vitro and in vivo. Mutants lacking functional Enok exhibited defects in the localization of Oskar (Osk) to the posterior end of the oocyte, resulting in loss of germline formation and abdominal segments in the embryo. RNA sequencing (RNA-seq) analysis revealed that spire (spir) and maelstrom (mael), both required for the posterior localization of Osk in the oocyte, were down-regulated in enok mutants. Chromatin immunoprecipitation showed that Enok is localized to and acetylates H3K23 at the spir and mael genes. Furthermore, Gal4-driven expression of spir in the germline can largely rescue the defective Osk localization in enok mutant ovaries. These results suggest that the Enok-mediated H3K23 acetylation (H3K23Ac) promotes the expression of spir, providing a specific mechanism linking oocyte polarization to histone modification (Huang, 2014).

This study reveals a previously unknown transcriptional role for Enok in regulating the polarized localization of Osk during oogenesis through promoting the expression of spir and mael. Spir and Mael are required for the properly polarized MT network in oocytes from stages 8 to 10A. However, protein levels of both decreased at later stages of oogenesis, allowing reorganization of the MT network and fast ooplasmic streaming. The persistent presence of Spir extending into stage 11 led to loss of ooplasmic streaming and resulted in female infertility. These findings suggest that the temporal regulation of spir expression is crucial for oogenesis, and, interestingly, Enok protein levels were also reduced in egg chambers during stages 10-13 compared with stages 1-9. While the stability of Spir or the translation of spir mRNA may also be a target for regulation, the results suggest that Enok is involved in the dynamic modulation of spir transcript. Furthermore, the results demonstrate the importance of Enok for expression of spir and mael in both ovaries and S2 cells, suggesting that Enok may play a similar role in other Spir- or Mael-dependent processes such as heart development (Huang, 2014).

Notably, Mael is also important for the piRNA-mediated silencing of transposons in germline cells. Mutations in genes involved in the piRNA pathway, including aub and armitage (armi), result in axis specification defects in oocytes as well as persistent DNA damage and checkpoint activation in germline cells. The activation of DNA damage signaling is suggested to cause axis specification defects in oocytes, as the disruption of Osk localization in piRNA pathway mutants can be suppressed by mutations in mei-41 or mnk, which encode ATR or checkpoint kinase 2, respectively. However, mutation in mnk cannot suppress the loss of posteriorly localized Osk in the mael mutant oocyte, indicating that the oocyte polarization defect in the mael mutant is independent of DNA damage signaling. Therefore, although the possibility that the piRNA pathway is affected in enok mutants due to down-regulation of mael cannot be excluded, the Osk localization defect in the enok mutant oocyte is likely independent of mei-41 and mnk (Huang, 2014).

In addition to the osk mRNA localization defect, both spir and mael mutants affect dorsal-ventral (D/V) axis formation in oocytes. However, no defects in the D/V patterning were observed in the eggshells of enok mutant germline clone embryos. Interestingly, among the spir mutant alleles that disrupt formation of germ plasm, only strong alleles result in dorsalized eggshells and embryos, while females with weak alleles produce eggs with normal D/V patterning. Since the enok¹ and enok² ovaries still express ~25% of the wild-type levels of spir mRNA, enok mutants may behave like weak spir mutants. Similarly, the ~40% reduction in mael mRNA levels in enok mutants as compared with the wild-type control may not have significant effects on the D/V axis specification (Huang, 2014).

Redundancy in HAT functions has been reported for both Moz and Sas3, the mammalian and yeast homologs of Enok, respectively. In yeast, deletion of either GCN5 (encoding the catalytic subunit of ADA and SAGA HAT complexes) or SAS3 is viable. However, simultaneously deleting GCN5 and SAS3 is lethal due to loss of the HAT activity of the two proteins, suggesting that Gcn5 and Sas3 can compensate for each other in acetylating histone residues. Indeed, while deleting SAS3 alone had no effect on the global levels of H3K9Ac and H3K14Ac, disrupting the HAT activity of Sas3 in the gcn5Δ background greatly reduced the bulk levels of H3K9Ac and H3K14Ac in yeast. Also, mammalian Moz targets H3K9 in vivo and regulates the expression of Hox genes, but the global H3K9Ac levels are not significantly affected in the homozygous Moz mutant, indicating that other HATs have overlapping substrate specificity with Moz. In flies, a previous study had reported that the H3K23Ac levels were reduced 35% in nejire (nej) mutant embryos, which lack functional CBP/p300. However, knocking down nej by dsRNA in S2 cells severely reduced levels of H3K27Ac but had no obvious effect on global levels of H3K23Ac. This study showed that the global H3K23Ac levels decreased 85% upon enok dsRNA treatment in S2 cells. This study also showed that the H3K23Ac levels are highly dependent on Enok in early and late embryos, larvae, adult follicle cells and nurse cells, and mature oocytes. Therefore, although Nej may also contribute to the acetylation of H3K23, the results indicate that, in contrast to its mammalian and yeast homologs, Enok uniquely functions as the major HAT for establishing the H3K23Ac mark in vivo (Huang, 2014).

The H3K23 residue has been shown to stabilize the interaction between H3K27me3 and the chromodomain of Polycomb. Therefore, acetylation of H3K23 may affect the recognition of H3K27me3 by the Polycomb complex. Another study showed that the plant homeodomain (PHD)-bromodomain of TRIM24, a coactivator for estrogen receptor α in humans, binds to unmodified H3K4 and acetylated H3K23 within the same H3 tail. Also, the levels of H3K23Ac at two ecdysone-inducible genes, Eip74EF and Eip75B, have been shown to correlate with the transcriptional activity of these two genes at the pupal stage, suggesting the involvement of H3K23Ac in ecdysone-induced transcriptional activation. This study further provided evidence for the activating role of the Enok-mediated H3K23Ac mark in transcriptional regulation (Huang, 2014).

In mammals, MOZ functions as a key regulator of hematopoiesis. Interestingly, one of the genes encoding mammalian homologs of Spir, spir-1, is expressed in the fetal liver and adult spleen, indicating the expression of spir-1 in hematopoietic cells. Thus, it will be intriguing to investigate whether the Drosophila Enok-Spir pathway is conserved in mammals and whether Spir-1 functions in hematopoiesis. Taken together, the results demonstrate that Enok functions as an H3K23 acetyltransferase and regulates Osk localization, linking polarization of the oocyte to histone modification (Huang, 2014).

Maelstrom represses canonical Polymerase II transcription within bi-directional piRNA clusters in Drosophila melanogaster

In Drosophila, 23-30 nt long PIWI-interacting RNAs (piRNAs) direct the protein Piwi to silence germline transposon transcription. Most germline piRNAs derive from dual-strand piRNA clusters, heterochromatic transposon graveyards that are transcribed from both genomic strands. These piRNA sources are marked by the heterochromatin protein 1 homolog Rhino (Rhi), which facilitates their promoter-independent transcription, suppresses splicing, and inhibits transcriptional termination. This study reports that the protein Maelstrom (Mael) represses canonical, promoter-dependent transcription in dual-strand clusters, allowing Rhi to initiate piRNA precursor transcription. Mael also represses promoter-dependent transcription at sites outside clusters. At some loci, Mael repression requires the piRNA pathway, while at others, piRNAs play no role. It is proposed that by repressing canonical transcription of individual transposon mRNAs, Mael helps Rhi drive non-canonical transcription of piRNA precursors without generating mRNAs encoding transposon proteins (Chang, 2018).

Fly piRNA clusters must solve a gene expression paradox. They record the ancient and contemporary exposure of the animal to transposon invasion, and this information must be copied into RNA in order to generate protective, anti-transposon piRNAs. However, recent transposon insertions retain the ability to produce mRNA encoding proteins required for their transposition. In flies, dual-strand piRNA clusters solve this paradox by using Rhi to initiate non-canonical transcription of unspliced RNA from both genomic strands, generating piRNA precursors, while repressing promoter-initiated, canonical transcription. These data suggest that Mael is required for this second process, allowing dual-strand piRNA clusters to safely generate piRNA precursor transcripts without risking production of transposon mRNAs. Within dual-strand clusters, Mael is likely guided to its targets by the piRNA pathway. However, the current analyses also predict that for some loci, Mael functions to repress canonical transcription in heterochromatin separately from the piRNA pathway, probably via one or more proteins that direct Mael to specific genomic sites (Chang, 2018).

In mael^M391/r20 ovaries, piRNAs mapping to dual-strand clusters decrease, despite a concomitant increase in canonical transcription from these same loci. The data suggest that in mael^M391 mutants, Rhi-mediated non-canonical transcription, cluster transcript export, and ping-pong amplification become uncoupled. Perhaps, canonical transcription and Rhi-mediated non-canonical transcription compete for Pol II. Instead of fueling piRNA production, the canonical transcripts from dual-strand piRNA clusters produced in the absence of Mael are translated into protein. Mael therefore contributes to dual-strand cluster piRNA production by tipping the balance toward non-canonical transcription (Chang, 2018).

The data indicate that in addition to relying on the piRNA pathway, Mael can also be guided to its targets by piRNA-independent mechanisms. Moreover, piRNA-dependent repression by Mael may be widespread outside of flies: although Drosophila melanogaster piwi is found only in the gonads, piwi is expressed broadly in the soma of most arthropods. In fact, 12 arthropods with somatic piRNAs also express mael in the soma, while 3 arthropods with no detectable piRNAs outside the gonads have low or undetectable somatic mael mRNA (Chang, 2018).

In male mice, loss of MAEL also leads to loss of piRNAs, germline transposon derepression, and sterility. As in flies, loss of MAEL in mice does not trigger loss of heterochromatin: DNA methylation of L1 elements is unchanged (Chang, 2018).

Because Mael is conserved from protists to humans, it is hypothesized that in different organisms Mael may be co-opted by different pathways to repress transcription of various targets. Prior studies suggest a model for how Mael confers repression. In protists, the MAEL domain was predicted to degrade RNA and may directly destroy nascent transcripts. In insects, the MAEL domain interacts with single-stranded RNA; it is speculated that fly Mael may have retained a role in destabilizing RNA. In this view, Mael may promote premature termination or degradation of nascent transcripts. In addition, because fly Mael has a partial HMG domain, it may also directly bind to DNA and repress transcription by preventing canonical core transcription factors from binding to promoters. Another possibility is that fly Mael may play a role in establishing or maintaining chromatin modifications not monitored in this study. Consistent with all of these possible mechanisms, non-canonical transcription mediated by Rhi is expected to be unaffected by Mael because the Rhi allows transcriptional initiation in dual-strand clusters without need for promoters and prevents degradation of unspliced piRNA precursor transcripts (Chang, 2018).

PROTEIN STRUCTURE

Amino Acids - 459

Structural Domains

Homologs of Maelstrom can be identified in mosquito (Anopheles gambiae), honey bee (Apis mellifera), mouse and human. A ClustalW alignment of the Drosophila, mosquito and human homologs shows 7.3% identity and 25.9% similarity shared between the three proteins. Fourth iteration Psi-blast searches with the Drosophila homolog gives overall E-values of e^-151 (to the mosquito homolog, agCP12344) and e^-150 (to the human homolog, FLJ14904). In addition, a partial potential HMG-box is found in Drosophila Maelstrom (residues 2-50), whereas a canonical HMG-box is found in the human homolog (residues 5-65) (Findley, 2003).

EVOLUTIONARY HOMOLOGS

Mouse maelstrom, a component of nuage, is essential for spermatogenesis and transposon repression in meiosis

Tight control of transposon activity is essential for the integrity of the germline. Recently, a germ-cell-specific organelle, nuage, was proposed to play a role in transposon repression. To test this hypothesis, a murine homolog of a Drosophila nuage protein Maelstrom was disrupted. Effects on male meiotic chromosome synapsis and derepression of transposable elements (TEs) were observed. In the adult Mael^-/- testes, LINE-1 (L1) derepression occurred at the onset of meiosis. As a result, Mael^-/- spermatocytes were flooded with L1 ribonucleoproteins (RNPs) that accumulated in large cytoplasmic enclaves and nuclei. Mael^-/- spermatocytes with nuclear L1 RNPs exhibited massive DNA damage and severe chromosome asynapsis even in the absence of SPO11-generated meiotic double-strand breaks. This study demonstrates that MAEL, a nuage component, is indispensable for the silencing of TEs and identifies the initiation of meiosis as an important step in TE control in the male germline (Soper, 2008).

These studies of Mael mutant testes suggest a close functional relationship of MAEL with MIWI2 and MILI, two of three murine PIWI-like proteins. Mael-, Mili-, and Miwi2-deficient spermatocytes fail to complete meiotic prophase and to assemble functional synaptonemal complexes. Interestingly Mael- and Miwi2-deficient spermatocytes develop identical atypical morphologies (not reported for the Mili mutant) prior to their elimination by apoptosis. Furthermore, Mael and Miwi2 mutant testes exhibit a common pattern of TE activation (high levels of L1 expression and only modest derepression of IAP) that contrasts TE expression in the Mili mutant. These data suggest that MAEL plays a more prominent role in the MIWI2-dependent processes, possibly at a post-piRNA production step (since no reduction of prepachytene piRNAs was observed in the Mael mutant). Given that MAEL was reported to shuttle between the nucleus and cytoplasm in Drosophila and MAEL complexes were observed in the nucleus and at nuclear pores in this study, it is speculated that MAEL may facilitate trafficking of MIWI2-piRNA complexes to or from nuage (Soper, 2008).

Of particular interest is the observation of L1 expression at the onset of meiosis in the absence of MAEL. It is generally accepted that de novo DNA methylation of TEs in gonocytes of late gestation male fetuses is crucial for TE repression in the male germline. In addition, recent studies demonstrated essential roles of MILI and MIWI2 in de novo DNA methylation of TEs in fetal gonocytes. Once established in the fetal germ cells, repressive DNA methylation of TEs is expected to be maintained in the male germline into the adulthood. One would expect, therefore, L1 derepression to be apparent in both premeiotic and meiotic germ cell populations of an adult Mael-deficient testis. In this study, however, L1 RNA and MAEL isoform ORF1p are found exclusively in the adult Mael^-/- meiotic germ cells and not in the spermatogonia. L1 ORF1p expression starts in preleptotene Mael^-/- spermatocytes (characterized by the premeiotic S phase) and persists until germ cell elimination. These results suggest a more dynamic control of TE expression at the onset of meiosis and parallel the findings of an earlier study of L1 expression in the testes. Taken together, the two studies suggest a tantalizing possibility of the existence of an epigenetic reprogramming step at the onset of meiosis that involves transient derepression of TEs (Soper, 2008).

This study provides compelling evidence of a highly detrimental effect of TE derepression on the male germline. L1 transcriptional derepression in the absence of MAEL leads to flooding of spermatocytes with ORF1p found both in the cytoplasm and nuclei. Cytoplasmic ORF1p is often present in large enclaves that contain vast numbers of 25-30 nm particles that could be identical to L1 RNPs observed previously. Nuclear ORF1p localization strongly correlates with accumulation of severe DNA damage that is mechanistically distinct from developmentally programmed SPO11-generated meiotic DSBs. The presence of numerous RAD51 foci in the absence of SPO11 further supports the idea that Mael^-/- spermatocytes harbor an endonuclease that is absent or inactive in wild-type and Spo11^-/- spermatocytes. A leading candidate for such an enzyme is L1-encoded ORF2p, whose N terminus has endonuclease activity that is required for retrotransposition and generation of DSBs in cultured human cell lines. Finally, nuclear accumulation of L1 RNPs also strongly correlates with defects in chromosome synapsis. A great majority of Mael^-/- spermatocytes exhibiting the most severe disruption of synapsis contain nuclear L1 RNPs (89% of cells in the univalent class). The precise mechanism of suppression of synapsis by L1 is yet to be understood, but the results already suggest a strong correlation between the frequency of nuclear L1 RNPs, the severity of DNA damage, and asynapsis. Cumulatively, this study vividly illustrates an absolute requirement for efficient TE restraining mechanisms in the mammalian germline (Soper, 2008).

date revised: 17 December 2021

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