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krimper: Biological Overview | References

PIWI proteins use guide piRNAs to repress selfish genomic elements, protecting the genomic integrity of gametes and ensuring the fertility of animal species. Efficient transposon repression depends on amplification of piRNA guides in the ping-pong cycle, which in Drosophila entails tight cooperation between two PIWI proteins, Aub and Ago3. This study shows that post-translational modification, symmetric dimethylarginine (sDMA), of Aub is essential for piRNA biogenesis, transposon silencing and fertility. Methylation is triggered by loading of a piRNA guide into Aub, which exposes its unstructured N-terminal region to the PRMT5 methylosome complex. Thus, sDMA modification is a signal that Aub is loaded with piRNA guide. Amplification of piRNA in the ping-pong cycle requires assembly of a tertiary complex scaffolded by Krimper, which simultaneously binds the N-terminal regions of Aub and Ago3. To promote generation of new piRNA, Krimper uses its two Tudor domains to bind Aub and Ago3 in opposite modification and piRNA-loading states. These results reveal that post-translational modifications in unstructured regions of PIWI proteins and their binding by Tudor domains that are capable of discriminating between modification states is essential for piRNA biogenesis and silencing (Huang, 2021).

The PIWI-interacting RNA (piRNA) pathway acts as a conserved defensive system that represses the proliferation of transposable elements (TEs) in the germline of sexually reproducing animals. Loss of PIWI proteins causes derepression of transposons associated with gametogenesis failure and sterility in flies and mice. PIWI proteins recognize transposon targets with help of the associated small (23-30 nt) non-coding RNA guides, piRNAs (Huang, 2021).

PIWI proteins belong to the conserved Argonaute protein family present in all domains of life. Argonautes bind nucleic acid guides and share common domain architecture, all containing the conserved N, PAZ, MID, and PIWI domains. The MID and PAZ domains bind the 5' and 3' ends of the guide RNA, respectively. The PIWI domain contains an RNase-H-like fold with a conserved amino acid tetrad that endows Argonautes with endonuclease activity for precise cleavage of the target. The degradation of complementary target mRNA by PIWI proteins can trigger the generation of new RNA guides in a process termed the ping-pong cycle. Ping-pong requires cooperativity between two PIWI molecules as the product resulting from target cleavage by one protein is passed to the other and is converted to a new piRNA guide. In Drosophila, two distinct cytoplasmic PIWI proteins, Aub and Ago3, cooperate in the ping-pong cycle with each protein generating an RNA guide that is loaded into its partner. Amplification of piRNA guides through the ping-pong cycle is believed to be essential for efficient transposon repression as it allows the pathway to mount an adaptive response to actively transcribed transposons (Huang, 2021).

In addition to four conserved domains, eukaryotic members of the Argonaute family, including PIWI proteins, contain an N-terminal extension region of various lengths with low sequence conservation. Structural studies of Agos suggest that the N-terminal regions adopt a disordered conformation. Despite low overall conservation, the N-terminal region of the majority of PIWI proteins harbors arginine-rich (A/G)R motifs. In both insects and mammals, these motifs were shown to be substrates for post-translational modification by the PRMT5 methyltransferase, which produces symmetrically dimethylated arginine (sDMA) residues. Loss of Prmt5 (encoded by the Csul and Vls genes) in Drosophila leads to reduced piRNA level and accumulation of transposon transcripts in germ cells, suggesting that sDMA modification of PIWIs plays an important role in the piRNA pathway (Huang, 2021).

Multiple Tudor domain-containing proteins (TDRDs) can bind to sDMA modifications. Aromatic residues in binding pocket of Tudor domains form cation-π interactions with sDMA. Studies in Drosophila and mouse revealed that several TDRDs interact with PIWIs and are involved in piRNA-guided transposon repression, although their specific molecular functions remain poorly understood. Previously it was found that the Tudor-domain containing protein Krimper is required for ping-pong piRNA amplification and is capable of both self-interactions and binding of the two ping-pong partners, Aub and Ago3. Krimper co-localizes with Aub and Ago3 in nuage, a membraneless perinuclear cytoplasmic compartment where piRNA-guided target degradation and ping-pong are proposed to take place. Ago3 requires Krimper for recruitment into this compartment, though Aub does not. These results led to the proposal that Krimper assembles a complex that brings Ago3 to Aub and coordinates ping-pong in nuage. However, the architecture of the ping-pong piRNA processing (4 P) complex and the extent to which Krimper regulates ping-pong remained unresolved (Huang, 2021).

Both the ping-pong cycle and sDMA modification of PIWI proteins are conserved features of the piRNA pathway, found in many organisms, suggesting that these processes are essential for pathway functions. sDMA modification of PIWIs provides a binding platform for interactions with Tudor-domain proteins, however, its biological function and regulation are not known. Despite the essential role of ping-pong in transposon repression, there is little understanding of the molecular mechanisms that control this process. This study revealed the biological function of Aub and Ago3 sDMA modifications and show that it plays an essential role in orchestrating assembly of the 4 P complex in the ping-pong cycle. The modification signals whether PIWI proteins are loaded with guide piRNA, and this information is used to assemble a ping-pong complex that is receptive for directional transfer of RNA to an unloaded PIWI protein (Huang, 2021).

Although PIWI proteins and piRNAs share many similarities with other Agos and their RNA guides, the piRNA pathway has evolved unique features that are essential for its function as an adaptive genome defense system. One such unique property is the amplification of piRNAs that target active transposons in the ping-pong cycle. Ping-pong employs the intrinsic RNA binding and processing capabilities of Ago proteins, however, it creates new functionality through the cooperation between two PIWI proteins. The results indicate that the ping-pong cycle and sDMA-modification are tightly linked and that the modification status of PIWI proteins regulates the assembly of the ping-pong processing complex (Huang, 2021).

Several lines of evidence suggest that sDMA modification of Aub is induced by the binding of a piRNA guide. First, Aub mutants that are deficient in piRNA binding due to mutation in either the RNA 5' or the 3' end binding pocket have a decreased level of sDMA modification. Second, disruption of piRNA biogenesis diminishes methylation of wild-type Aub. Finally, the loading of chemically synthesized RNA into Aub promotes its association with the methylosome complex and sDMA modification. In contrast, sDMA modification of Aub is not required for its loading with piRNA and for its slicer activity. Together, these results suggest that sDMA modification of Aub acts as a signal of its piRNA-bound state (Huang, 2021).

The results suggest that piRNA loading induces sDMA methylation through a conformational change that makes the N-terminal sequence accessible to the methylation enzyme. While unloaded Aub is poorly methylated, the N-terminal sequence alone is a good substrate for methylation. Insertion of a sequence between the N-terminal region and the rest of the protein also promotes methylation (despite the protein not being able to bind piRNA), suggesting that other parts of the protein inhibit modification. Finally, partial proteolysis indicates that Aub undergoes a conformation change upon piRNA loading. Combined, these experiments suggest that the N-terminal sequence is poorly accessible to the modifying enzyme until Aub binds a guide RNA, inducing a conformation change that exposes its N-terminus (Huang, 2021).

Structural differences between empty and loaded states were reported for several prokaryotic and eukaryotic Agos, corroborating the idea that binding to guide RNA induces conformational change. The PAZ domain of Agos exhibit a high level of flexibility upon loading of guide RNA/DNA. During the recognition of target RNA, the PAZ domain undergoes a conformational transition that releases the 3' end of the guide and facilitates downstream guide-target base pairing. The results indicate that binding of the 3' end by the PAZ domain is critical for sDMA modification of Aub's N-terminal region. Unfortunately, the N-terminal extension region was often truncated to facilitate Ago expression and crystallization and thus reported structures do not provide information about the N-terminal extension region. If the N-terminal region is preserved, it exists in an unstructured conformation that remains unresolved by crystallography. However, piRNA loading of the nuclear PIWI protein in Drosophila was shown to induce a conformational change that exposes the nuclear localization sequence (NLS) located in its N-terminus and to enable its binding to importin. Thus, two PIWI clade proteins, Aub and Piwi, harbor an N-terminal sequence that becomes accessible upon piRNA loading and its exposure promotes interactions with other factors and regulates protein function. Similar to Aub, the N-terminal extension region of Ago3 also harbors a (G/A)R motif that can be modified. Considering that piRNA binding triggers exposure of the N-terminus in Aub and Piwi, a similar process might occur in Ago3. Indeed, previous studies and the current results revealed that, unlike the bulk of the cellular Ago3 pool, Krimper-bound Ago3 is both unloaded and unmethylated, indicating that piRNA binding and modification are correlated for Ago3 as well as for Aub (Huang, 2021).

The results demonstrate the importance of the N-terminal region in the function of PIWI proteins. Unlike other domains (PAZ, MID, PIWI) of Argonautes with well-characterized functions in RNA guide binding and target cleavage, the N-terminal region has received little attention due to its disordered conformation and its low conservation between different Agos. The results suggest that the low conservation and absence of a fixed structure are in fact important features of the N-terminal region that are critical for PIWI proteins function. The flexible structure of this region might provide sensitivity to changes in overall protein conformation, such as the changes triggered by guide RNA binding. In Aub and Ago3, the modification and binding of sDMA sites to other proteins, as well as NLS-mediated interaction of Piwi require only a short linear motif, and thus the N-terminal region does not require a strongly conserved sequence or rigid folding. In agreement with this, the presence of a (G/A)R motif in Aub and Ago3 proteins is conserved in other Drosophila species, however, the specific position and sequence context of the motif is diverse. The poor similarity between N-terminal sequences of different Agos might endow them with distinct functions. It might be worth exploring whether signaling of the guide-loading state through exposure of the N-terminal region is also conserved in Ago-clade proteins and whether it regulates their function (Huang, 2021).

The central feature of ping-pong is that the cleavage of target RNA by one PIWI protein results in the transfer of the cleaved product to another PIWI protein. Although the original model of ping-pong did not provide information on the molecular complex and interactions within the complex, ping-pong intuitively implies physical proximity between the two PIWI proteins followed by complex molecular rearrangements. This study found that, although sDMA modification does not affect slicer activity of Aub, information about the piRNA-loading state of PIWI proteins signaled by their sDMA modifications is used to assemble a complex that enables the transfer of the processed RNA from Aub to Ago3 (Huang, 2021).

While previous findings strongly suggest that Krimper plays a role in the assembly of the ping-pong piRNA processing (4 P) complex in which Aub and Ago3 are brought into close physical proximity (Webster, 2015), the architecture of this complex and the extent to which Krimper regulates ping-pong remained unknown. The current results indicate that a single Krimper molecule interacts simultaneously with Aub and Ago3, suggesting that ping-pong takes place within a tertiary complex containing one molecule of each protein. Krimper actively selects the two ping-pong partners using the distinct specificities of its two Tudor domains: eTud1 uniquely binds Ago3, while eTud2 recognizes modified Aub. This study found that in vitro the eTud2 domain is capable of binding both sDMA-modified Aub and Ago3 peptides, however, in vivo Krimper complexes were reported to contain exclusively unmodified Ago3, suggesting that in the proper cellular context eTud2 only binds sDMA-Aub. Thus, the domain architecture of Krimper ensures that tertiary complexes contain Aub-Ago3 partners rather than random pairs. This finding is in line with the observation that ping-pong occurs predominantly between Aub and Ago3, although, in principle, ping-pong can take place between two identical proteins, and a small level of homotypic Aub/Aub ping-pong was previously detected. Thus, these results suggest that the propensity for heterotypic ping-pong is, at least in part, due to Krimper (Huang, 2021).

Ping-pong not only requires the physical proximity of two PIWI proteins but also that they have opposite piRNA-loading states: one protein induces piRNA-guided RNA cleavage (and therefore has to be loaded with a piRNA guide), while the other accepts the product of this reaction (and therefore has to be free of piRNA). The results suggest that the opposite binding preference of the two Tudor domains towards sDMA ensures that the tertiary complex contains PIWI proteins in opposite RNA-loading states. While the overall fold structure of the two Tudor domains is similar, they have critical differences responsible for their distinct binding preferences. The binding pocket of eTud2 is similar to that of other Tudor domains and contains four aromatic residues that interact with sDMA. As sDMA modification of Aub signals its piRNA-binding status, the binding of eTud2 to modified Aub ensures that the complex contains Aub/piRNA. The structural studies and in vitro binding assays revealed that Ago3 binds to eTud1 in its unmethylated state and sDMA modification of any of the Arg residues within its (A/G)R motif prevents this interaction. The unusual binding preference of eTud1 is reflected in its non-canonical binding pocket, which lacks three of the four conserved aromatic residues. The binding of methylated Aub and unmethylated Ago3 ensures that Aub has a guide piRNA and Ago3 is free, thus enabling loading of Ago3 with RNA generated by Aub/piRNA-induced cleavage (Huang, 2021).

The architecture of the tertiary complex assembled by Krimper permits Aub-dependent generation and loading of RNA into Ago3. However, the ping-pong cycle also includes the opposite step, Ago3-dependent generation of Aub piRNA (henceforth these steps were termed 'ping' and 'pong'). The results suggest that the ping and pong steps require the assembly of two distinct complexes discriminated by the modification status of Aub and Ago3 (Huang, 2021).

As a single Krimper simultaneously binds Aub and Ago3, Krimper dimerization might be dispensable for ping-pong, raising the question of what the function of Krimper self-interaction is. Previous findings suggest that Krimper forms a scaffold for assembly of nuage, a membraneless organelle (MLO) that surrounds nuclei of nurse cells and resembles other MLO possibly formed through liquid-liquid phase separation. Several lines of evidence point at Krimper as an essential component of nuage that acts as a scaffold for its assembly and the recruitment of client components. First, unlike other nuage components, FRAP measurements show very little Krimper exchange between nuage and the dispersed cytoplasmic compartment. Second, wild-type, but not mutant Krimper that lacks the self-interaction domain, forms cytoplasmic granules upon expression in heterologous cells that do not contain other nuage proteins. In contrast, other nuage components including Aub and Ago3 are dispersed in the cytoplasm when expressed in a similar setting, suggesting that they do not form condensates on their own and rely on other components for recruitment to nuage. Krimper recruits both Aub and Ago3 into MLO that it forms in heterologous cells. Combined, these data indicating that Krimper works as a scaffold, and Ago3 and Aub as its clients for nuage assembly. Thus, the interactions between Krimper and the N-terminal regions of Aub and Ago3 is not only essential for the assembly of the tertiary molecular complex but is also responsible for the recruitment of these proteins into membraneless cellular compartment (see Model for sDMA regulation and its function in ping-pong cycle). The high local concentration of proteins and RNA involved in the piRNA pathway in nuage might enhance the efficiency of ping-pong as well as the recognition of RNA targets by Aub and Ago3 (Huang, 2021).

Drosophila Interspecific Hybridization Causes A Deregulation of the piRNA Pathway Genes

Almost all eukaryotes have transposable elements (TEs) against which they have developed defense mechanisms. In the Drosophila germline, the main transposable element (TE) regulation pathway is mediated by specific Piwi-interacting small RNAs (piRNAs). Nonetheless, for unknown reasons, TEs sometimes escape cellular control during interspecific hybridization processes. Because the piRNA pathway genes are involved in piRNA biogenesis and TE control, nine key genes from this pathway were sequenced and characterized in Drosophila buzzatii and Drosophila koepferae species, and their expression pattern in ovaries of both species and their F1 hybrids was studied. It was found that gene structure is, in general, maintained between both species and that two genes-armitage and aubergine-are under positive selection. Three genes-krimper, methyltransferase 2, and zucchini-displayed higher expression values in hybrids than both parental species, while others had RNA levels similar to the parental species with the highest expression. This suggests that the overexpression of some piRNA pathway genes can be a primary response to hybrid stress. Therefore, these results reinforce the hypothesis that TE deregulation may be due to the protein incompatibility caused by the rapid evolution of these genes, leading to a TE silencing failure, rather than to an underexpression of piRNA pathway genes (Gamez-Visairas, 2020).

Aub and Ago3 Are Recruited to Nuage through Two Mechanisms to Form a Ping-Pong Complex Assembled by Krimper

In Drosophila, two Piwi proteins, Aubergine (Aub) and Argonaute-3 (Ago3), localize to perinuclear 'nuage' granules and use guide piRNAs to target and destroy transposable element transcripts. This study finds that Aub and Ago3 are recruited to nuage by two different mechanisms. Aub requires a piRNA guide for nuage recruitment, indicating that its localization depends on recognition of RNA targets. Ago3 is recruited to nuage independently of a piRNA cargo and relies on interaction with Krimper, a stable component of nuage that is able to aggregate in the absence of other nuage proteins. This study shows that Krimper interacts directly with Aub and Ago3 to coordinate the assembly of the ping-pong piRNA processing (4P) complex. Symmetrical dimethylated arginines are required for Aub to interact with Krimper, but they are dispensable for Ago3 to bind Krimper. This study reveals a multi-step process responsible for the assembly and function of nuage complexes in piRNA-guided transposon repression (Webster, 2015).

The interaction between Aub and Ago3 lies at the heart of the ping-pong piRNA processing model; however, the molecular mechanism of this process remained poorly understood. No direct association between Aub and Ago3 was ever reported, which suggested that if Aub and Ago3 form a complex, this interaction is likely transient and mediated by other proteins. Genetic studies identified several genes implicated in the ping-pong process. The molecular function of these genes remained largely unknown: some of the encoded proteins might promote physical interactions between Aub and Ago3, while others might be working in other steps of ping-pong processing. The protein that promotes assembly of the ping-pong complex is expected to fulfill several criteria. First, this protein must directly interact with both Aub and Ago3. Second, it must be able to form a complex that contains Aub and Ago3 simultaneously, where Aub and Ago3 must be in a state that is compatible with progression of the ping-pong cycle (i.e., if Aub is loaded with piRNA then Ago3 should be unloaded). Third, mutation of the corresponding gene for this protein should lead to disruption of ping-pong piRNA processing. On a cellular level the protein is expected to co-localize with both Aub and Ago3. Finally, it might be expected to be a stable component of nuage that is capable of recruiting one or both Piwi proteins to this compartment (Webster, 2015).

According to the results of this study, Krimp fulfills all the criteria as a factor that promotes the assembly of the 4P complex. First, Krimp directly interacts with two ping-pong partners, Aub and Ago3. Interestingly, Krimp uses different mechanisms to bind each Piwi proteins: binding to Aub depends on methylation of N-terminal arginine residues, while binding to Ago3 is independent of sDMA. Although it was not possible to directly compare the strength of binding between Krimp and the two Piwi proteins, the pattern of subcellular localization of the three proteins in germ cells suggests that Krimp predominantly binds Ago3 in a stable complex, while interactions with Aub are transient. Due to the ability of Krimp to aggregate, Krimp dimers can simultaneously interact with Aub and Ago3, allowing for the formation of transient Aub/Krimp/Krimp/Ago3 complex. Importantly, Ago3 associates with Krimp in its unloaded state, indicating that it is ready to receive a substrate from Aub cleavage. Krimp mutation disrupts heterotypic (Aub-Ago3) and homotypic (Aub-Aub) ping-pong]. Krimp has low mobility in nuage and is able to form granules by itself in the absence of other nuage proteins. Krimp is indispensable for recruitment of Ago3 to nuage, and although it is not required for recruitment of Aub per se, it promotes concentration of Aub in prominent nuage granules together with Ago3. Overall the data indicate that Krimp is a stable component of nuage that recruits unloaded Ago3 into this cellular compartment. The Krimp-Aub interaction assembles the 4P complex that coordinates the passage of cleaved RNA fragments from Aub to unloaded Ago3, defining the essential step in the ping-pong cycle. The 4P complex seems to be transient and disassembles rapidly upon loading of Ago3 with secondary piRNA. While the ping-pong mechanism is conserved in mouse, there is no direct ortholog of Krimp in mammals. However, several proteins with one or more Tudor domains are implicated in the piRNA pathway and TE repression in mouse. It is possible that one or more Tudor-domain proteins that are able to aggregate and interact with Piwi proteins could play a role that is analogous to Krimp in flies (Webster, 2015).

Previously, Qin/Kumo was reported to mediate the interaction between Aub and Ago3 and it fulfills many criteria expected of a component responsible for the assembly of the ping-pong complex. RNA helicase Vasa was also proposed to coordinate the assembly of the ping-pong (Amplifier) complex in silkmoth cells. Vasa might be involved in promoting transition of intermediate ping-pong complex to the next step by unwinding piRNA from target RNA after cleavage by Aub. In the future, it will be important to determine how Krimp, Qin/Kumo, and Vasa cooperate in the ping-pong pathway (Webster, 2015).

Previous studies showed that Aub, Ago3, and several other proteins that are required for piRNA repression localize to the distinct subcellular compartment: perinuclear nuage granules. Furthermore, several mutations that disrupt nuage also cause failure in piRNA biogenesis and result in derepression of TEs. These observations led to the hypothesis that nuage is the subcellular compartment where piRNA processing, target recognition, and repression take place. The mechanism by which nuage components assemble and recruit Piwi proteins remained unknown. Surprisingly, it was found that Aub and Ago3 are recruited to nuage through different molecular mechanisms. The main factor determining Aub localization to nuage is its ability to bind a piRNA guide. The requirement of piRNA for Aub localization in nuage helps to explain the results of previous studies, which found that deficiencies in nuclear proteins Rhino, UAP56, and Cutoff, vital for early steps of piRNA biogenesis, cause delocalization of Aub from nuage. The dependence on a piRNA guide for Aub localization suggests that recognition of target RNA molecules might tether Aub in nuage. Alternatively, binding of piRNA might lead to conformational changes in Aub that promote its interaction with other proteins in nuage (Webster, 2015).

The results show that nuage is a heterogenous compartment that can be further subdivided into two structurally and functionally different sub-compartments: one predominantly containing Aub alone and another where Aub and Ago3 co-localize. Aub covers the entire nuclear periphery, the compartment called Aub-nuage, while Ago3 localizes to a few distinct granules embedded in the smooth Aub-nuage. To effectively cleave transposon transcripts, Aub must scan all RNA transcripts exiting the nucleus. Therefore, it is proposed that smooth Aub-nuage is a compartment where cleavage of TE transcripts takes place. It is important to note that this step alone should be sufficient to repress TEs without the need of the ping-pong mechanism (Webster, 2015).

Accordingly, the majority of Aub molecules might not be involved in ping-pong interactions with Ago3 but will instead dissociate from nuage after successful cleavage of TE targets transcripts. Yet, some Aub complexes proceed to assemble transient complexes with Ago3 and Krimp to form Aub-Ago3 nuage granules that occupy a territory distinct from Aub-nuage. Assembly of the transient Aub/Krimp/Ago3 4P complex provides an opportunity for the ping-pong cycle to take effect. Thus, in contrast to smooth Aub-nuage, the function of the distinct Ago3/Aub granules is to provide a subcellular compartment for ping-pong piRNA processing by accommodation of all players in one place. Together, these data can be integrated into a dynamic model that explains the existence of two distinct nuage compartments, the forces that drive recruitment of Piwi proteins to nuage and the function of Aub and Ago3 at the different steps of the piRNA pathway (Webster, 2015).

Krimper enforces an antisense bias on piRNA pools by binding AGO3 in the Drosophila germline

Piwi-interacting RNAs (piRNAs) suppress transposon activity in animal germ cells. In the Drosophila ovary, primary Aubergine (Aub)-bound antisense piRNAs initiate the ping-pong cycle to produce secondary AGO3-bound sense piRNAs. This increases the number of secondary Aub-bound antisense piRNAs that can act to destroy transposon mRNAs. This study shows that Krimper (Krimp), a Tudor-domain protein, directly interacts with piRNA-free AGO3 to promote symmetrical dimethylarginine (sDMA) modification, ensuring sense piRNA-loading onto sDMA-modified AGO3. In aub mutant ovaries, AGO3 associates with ping-pong signature piRNAs, suggesting AGO3's compatibility with primary piRNA loading. Krimp sequesters ectopically expressed AGO3 within Krimp bodies in cultured ovarian somatic cells (OSCs), in which only the primary piRNA pathway operates. Upon krimp-RNAi in OSCs, AGO3 loads with piRNAs, further showing the capacity of AGO3 for primary piRNA loading. It is proposed that Krimp enforces an antisense bias on piRNA pools by binding AGO3 and blocking its access to primary piRNAs (Sato, 2015).

The data suggest that Krimp regulates sDMA modification of AGO3 through direct binding to the N-terminal portion of the protein, thereby controlling interactions of other Tudor-domain proteins, such as Tud (see Krimp Enforces an Antisense Bias on piRNA Pools by Assembling AGO3 in the Ping-Pong Cycle) (Sato, 2015).

A recent study showed that a lack of Qin, another Tudor domain protein, disrupts the interaction between AGO3 and Aub and triggers Aub-Aub homotypic ping-pong in fly ovaries, resulting in an increase in the abundance of sense piRNAs while the overall abundance of piRNAs is preserved. Therefore, Qin seems to act to suppress Aub-Aub homotypic ping-pong by promoting the Aub-AGO3 interaction, which leads to an increase in heterotypic Aub-AGO3 ping-pong. A considerable amount of Aub-Aub homotypic ping-pong occurs even in controls, so Aub-Aub homotypic ping-pong is probably a default pathway for piRNA biogenesis; however, this pathway increases the proportion of sense piRNAs in the piRNA pool and so does not efficiently repress TEs. Therefore, there should be a system(s) that promotes heterotypic Aub-AGO3 ping-pong and that also increases the proportion of antisense piRNAs. Although Aub-Aub homotypic ping-pong also prevails in krimp mutant ovaries, the overall abundance of piRNAs in germline cells is dramatically decreased. This profile of piRNAs in krimp mutant ovaries is very similar to that in ago3 mutant ovaries in which the ping-pong cycle collapses. The increase in Aub-Aub homotypic ping-pong observed in krimp mutant ovaries may largely reflect the loss of AGO3-Aub heterotypic ping-pong, rather than a direct effect on promoting Aub-Aub homotypic ping-pong. Therefore, Krimp plays a very different role to Qin in the ping-pong cycle (Sato, 2015).

The ping-pong cycle is mediated by the Slicer-dependent mutual cleavage of sense and antisense transcripts of a TE, which should result in equal amounts of sense and antisense piRNAs. Thus, an important question is this: what makes sense piRNAs less abundant and disproportionately bound to AGO3 in the ping-pong cycle, enforcing the characteristic antisense bias of Aub-bound piRNAs? In other words, why is Aub-Aub homotypic ping-pong not sufficient to produce the characteristic antisense bias of Aub-bound piRNAs? In krimp mutant ovaries, AGO3 is no longer sDMA modified and is free from piRNAs. Therefore, Krimp not only masks the N-terminal portion of AGO3 where sDMA-modifiable arginine residues reside, but also mediates the sDMA modification that leads to interactions of AGO3 with other Tudor domain proteins, including Tud, and to its nuage localization. In this way, Krimp promotes the ping-pong cycle. Because sDMA modification of Aub is not affected in krimp mutant ovaries, complex formation between Aub loaded with primary piRNAs and Tudor domain proteins may occur before sDMA-modified AGO3 joins the complexes to ensure Aub-AGO3 heterotypic ping-pong. Qin may act in this step to suppress Aub-Aub homotypic ping-pong to further promote Aub-AGO3 heterotypic ping-pong (Sato, 2015).

AGO3 is clearly compatible with the primary piRNA pathway in OSCs when Krimp is depleted. Therefore, Krimp may not only promote the Aub-AGO3 heterotypic ping-pong cycle but also actively prevent AGO3 from becoming loaded with primary piRNAs in germline cells. In contrast, in aub mutant ovaries where AGO3 no longer accumulates at nuage but is sequestered into Krimp bodies, AGO3 is still loaded with reduced levels of piRNAs that have characteristics of primary piRNAs. These results suggest that complete blockage of primary piRNA loading onto AGO3 in ovarian germline cells requires an additional factor(s). An alternative possibility may be that AGO3 does not selectively accept primary piRNAs when aub is mutated but is perhaps loaded in a rather non-specific manner. Thus, together, these findings suggest that the characteristic antisense bias of Aub-bound piRNAs is created by the sum of piRNAs produced in the ping-pong cycle and the continual flow of primary piRNAs onto Aub. Aub is much more abundant than AGO3 in fly ovaries, further suggesting that the continuous flow of primary piRNAs to Aub should contribute to the antisense bias of Aub-bound piRNAs. Alternatively, it is also conceivable that AGO3-sense piRNA complexes may be catalytically more active than Aub-antisense piRNA complexes, potentially resulting in the antisense bias of Aub-bound piRNAs. However, in OSCs, AGO3 is loaded with primary piRNAs when Krimp is depleted while AGO3 in germline cells is not loaded with piRNAs. This suggests that in addition to Krimp, a second/backup system may also operate to further prevent AGO3 from associating with primary piRNAs in germline cells (Sato, 2015).

In aub mutant ovaries, Krimp is no longer accumulated at the nuage but forms Krimp bodies (Lim, 2007, Nagao, 2011), suggesting that the nuage localization of Krimp is Aub dependent. However, Krimp does not directly interact with Aub, and Krimp in OSCs forms Krimp bodies when Aub is ectopically expressed. Therefore, there must be a germline-specific factor(s) that links Aub and Krimp. The N-terminal coiled-coil domains of Krimp are required for Krimp body formation. Thus, a factor(s) may exist to mask the coiled-coil domains to prevent Krimp from forming the aggregates in germline cells. The function and/or stability of this putative factor could be Aub dependent in germline cells. The next key challenge will be to identify such a factor that regulates Krimp, thereby contributing to the operation of the ping-pong cycle (Sato, 2015).

Both AGO3 and Aub have the potential to load sense and antisense piRNAs, and the loading of these two proteins likely relies on the functions of interacting proteins, such as Krimp. It was therefore hypothesized that AGO3 and Aub are functionally very similar and that their interacting partners play essential roles in determining the behaviors of the two PIWI proteins, such that they effectively participate in ping-pong amplification. If both Aub and AGO3 are compatible with the primary piRNA pathway, why then are primary piRNAs loaded only onto Aub and Piwi? Perhaps this is a system to ensure a heterotypic ping-pong between Aub and AGO3 and some other as yet unknown mechanism to amplify antisense piRNAs. During Drosophila germline development, Piwi and Aub, but not AGO3, are directly deposited from mother to offspring through germline transmission. Antisense piRNAs that can act to repress the activation of TEs and initiate the ping-pong cycle are loaded onto Piwi and Aub. The maternal loading of antisense piRNAs, together with the functions of Tudor domain proteins such as Krimp and Qin, could establish an antisense bias in the ping-pong amplification loop (Sato, 2015).

There is no apparent homolog of Krimp in mammals. This may be because the ping-pong amplification mechanism in mammals is different from that in Drosophila. For example, the mouse PIWI protein, MILI, loads sense rather than antisense primary piRNAs. However, heterotypic ping-pong with ping-pong partner MIWI2 may not operate because, once loaded with secondary piRNAs, MIWI2 is imported into the nucleus to direct specific DNA methylation of transposon loci. It is likely that a Krimp-like protein exists in mammals to enforce a piRNA strand bias (Sato, 2015).

Gender-Specific Hierarchy in Nuage Localization of PIWI-Interacting RNA Factors in Drosophila

PIWI-interacting RNAs (piRNAs) are germline-specific small non-coding RNAs that form piRNA-induced silencing complexes (piRISCs) by associating with PIWI proteins, a subclade of the Argonaute proteins predominantly expressed in the germline. piRISCs protect the integrity of the germline genome from invasive transposable DNA elements by silencing them. Multiple piRNA biogenesis factors have been identified in Drosophila. The majority of piRNA factors are localized in the nuage, electron-dense non-membranous cytoplasmic structures located in the perinuclear regions of germ cells. Thus, piRNA biogenesis is thought to occur in the nuage in germ cells. Immunofluorescence analyses of ovaries from piRNA factor mutants have revealed a localization hierarchy of piRNA factors in female nuage. However, whether this hierarchy is female-specific or can also be applied in male gonads remains undetermined. This study shows by immunostaining of both ovaries and testes from piRNA factor mutants that the molecular hierarchy of piRNA factors shows gender-specificity, especially for Krimper (Krimp), a Tudor-domain-containing protein of unknown function(s): Krimp is dispensable for PIWI protein Aubergine (Aub) nuage localization in ovaries but Krimp and Aub require each other for their proper nuage localization in testes. This suggests that the functional requirement of Krimp in piRNA biogenesis may be different in male and female gonads (Nagao, 2011).

Unique germ-line organelle, nuage, functions to repress selfish genetic elements in Drosophila melanogaster

The nuage is an electron-dense perinuclear structure that is known to be a hallmark of animal germ-line cells. Although the conservation of the nuage throughout evolution accentuates its essentiality, its role(s) and the exact mechanism(s) by which it functions in the germ line still remain unknown. This paper reports a nuage component, Krimper (KRIMP), in Drosophila melanogaster and shows that it ensures the repression of the selfish genetic elements in the female germ line. The Krimp loss-of-function allele exhibited female sterility, defects in karyosome formation and oocyte polarity, and precocious osk translation. These phenotypes are commonly observed in the other nuage component mutants, vasa (vas) and maelstrom (mael), and the RNA-silencing component mutants, spindle-E (spn-E) and aubergine (aub), suggesting a shared underlying defect that uses RNA silencing. Moreover, it was demonstrated that the localization of the nuage components depends on both SPN-E and AUB and that the selfish genetic elements were derepressed to different extents in the nuage component mutants, as well as in aub and armitage (armi) mutants. In the nuage component mutants, vas, krimp, and mael, the levels of roo, I-element, and HeT-A repeat-associated small interfering RNAs were greatly reduced. Hence, these data suggest that the nuage functions as a specialized center that protects the genome in the germ-line cells via gene regulation mediated by repeat-associated small interfering RNAs (Lim, 2007).

Search PubMed for articles about Drosophila Krimper

Gamez-Visairas, V., Romero-Soriano, V., Marti-Carreras, J., Segarra-Carrillo, E. and Garcia Guerreiro, M. P. (2020). Drosophila Interspecific Hybridization Causes A Deregulation of the piRNA Pathway Genes. Genes (Basel) 11(2). PubMed ID: 32092860

Huang, X., Hu, H., Webster, A., Zou, F., Du, J., Patel, D. J., Sachidanandam, R., Toth, K. F., Aravin, A. A. and Li, S. (2021). Binding of guide piRNA triggers methylation of the unstructured N-terminal region of Aub leading to assembly of the piRNA amplification complex. Nat Commun 12(1): 4061. PubMed ID: 34210982

Lim, A. K. and Kai, T. (2007). Unique germ-line organelle, nuage, functions to repress selfish genetic elements in Drosophila melanogaster. Proc Natl Acad Sci U S A 104(16): 6714-6719. PubMed ID: 17428915

Nagao, A., Sato, K., Nishida, K. M., Siomi, H. and Siomi, M. C. (2011). Gender-Specific Hierarchy in Nuage Localization of PIWI-Interacting RNA Factors in Drosophila. Front Genet 2: 55. PubMed ID: 22303351

Sato, K., Iwasaki, Y. W., Shibuya, A., Carninci, P., Tsuchizawa, Y., Ishizu, H., Siomi, M. C. and Siomi, H. (2015). Krimper enforces an antisense bias on piRNA pools by binding AGO3 in the Drosophila germline. Mol Cell 59: 553-563. PubMed ID: 26212455

Webster, A., Li, S., Hur, J. K., Wachsmuth, M., Bois, J. S., Perkins, E. M., Patel, D. J. and Aravin, A. A. (2015). Aub and Ago3 Are Recruited to Nuage through Two Mechanisms to Form a Ping-Pong Complex Assembled by Krimper. Mol Cell 59(4): 564-575. PubMed ID: 26295961

date revised: 9 November 2021

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