InteractiveFly: GeneBrief

tejas: Biological Overview | References

PIWI-interacting RNAs (piRNAs), which protect genome from the attack by transposons, are produced and amplified in membraneless granules called nuage. In Drosophila, PIWI family proteins, Tudor-domain-containing (Tdrd) proteins, and RNA helicases are assembled and form nuage to ensure piRNA production. However, the molecular functions of the Tdrd protein Tejas (Tej) in piRNA biogenesis remain unknown. This study conducted a detailed analysis of the subcellular localization of fluorescently tagged nuage proteins and behavior of piRNA precursors. The results demonstrate that Tej functions as a core component that recruits Vasa (Vas) and Spindle-E (Spn-E) into nuage granules through distinct motifs, thereby assembling nuage and engaging precursors for further processing. This study also reveals that the low-complexity region of Tej regulates the mobility of Vas. Based on these results, it is proposed that Tej plays a pivotal role in piRNA precursor processing by assembling Vas and Spn-E into nuage and modulating the mobility of nuage components (Lin, 2023).

Transposons (transposable elements, TEs) are mobile genetic elements that exist in the genomes of all eukaryotic organisms and they occupy a substantial portion of genomes. They directly impair genomes by causing double-strand breaks, promoting ectopic recombination, and abolishing gene expression. PIWI-interacting RNAs (piRNAs), a class of 23-29-nt gonad-specific small RNAs, protect genome integrity by mitigating any catastrophes in germline cells that will be transmitted to the next generations. piRNAs are quite conserved and widely found among animals, and the model animal system, Drosophila, has been used to investigate and dissect the molecular mechanisms of piRNAs (Lin, 2023).

Drosophila piRNAs are processed from long piRNA precursor transcripts derived from genomic loci called piRNA clusters, where inactive or fragmented transposons are deposited. Discrete piRNA clusters are active in gonads, where they produce dual-strand piRNA precursors in germline cells or unistrand piRNA precursors in somatic gonadal cells. In germline cells, nascent piRNA precursors are transported to a unique, germline-specific membraneless structure called nuage in the perinuclear region via the Nxf3-Nxt1 pathway. Nuage consists of precursors and transposon RNAs being processed, two PIWI family proteins-Aub and Ago3-and other relevant components, DEAD-box RNA helicase Vasa (Vas), DEAH box helicase RNA helicase Spindle-E (Spn-E), and a group of Tudor domain-containing proteins (Tdrds), Krimper (Krimp), Tejas (Tej), Tudor, Tapas (Tap), Qin/Kumo, and Vreteno. After loading long piRNA precursors and transposon RNAs onto Aub and Ago3, they are cleaved and sliced into mature piRNAs, leading to the formation of antisense and sense piRNAs with a 10-nt complementarity. These processed piRNAs are further amplified in nuage in a feed-forward amplification cycle called the ping-pong cycle. However, the molecular mechanisms of nuage assembly are still unclear (Lin, 2023).

Although Tdrds are multifunctional, their overall activities are not fully understood. They interact with symmetrically demethylated arginine (sDMA), which is usually present at the N-terminus of PIWI family proteins, through the Tudor domain, thereby promoting aggregate formation in mammalian cells. This behavior implies the importance of molecular associations of Tdrds for nuage formation. Membraneless organelles composed of RNA and proteins are responsible for diverse RNA processing, including P-body and Yb body in Drosophila, which modulate the molecular organization in a process called phase separation. Two Tdrds localized in Drosophila nuage-Tej and Tap-contain an extended Tudor domain (eTudor) and an additional Lotus domain that is conserved from bacteria to eukaryotes. The Lotus domain was previously reported to interact with Vas, which is required for the piRNA pathway (Lin, 2023).

Of these two proteins, Tej/Tdrd5 is one of the key factors in the piRNA pathway in both Drosophila and mice (Patil, 2014; Patil, 2010; Yabuta, 2011). piRNAs are massively reduced with the displacement of other components from nuage in the absence of Tej/Tdrd5; however, the molecular functions of Tej remain elusive. This study identified the domains of Tej that interact with Vas and Spn-E, which are required for proper nuage formation and piRNA precursor processing, in addition to the contribution of the intrinsically disordered region (IDR) to the dynamics of other nuage components. It is proposed that Tej plays a pivotal role in piRNA precursor processing by recruiting Vas and Spn-E for nuage and modulating their dynamics for nuage assembly (Lin, 2023).

The piRNAs in Drosophila germline cells are produced and amplified in the membraneless organelle, nuage, which is assembled by orderly recruitment of the corresponding components to ensure its proper function. Although its precise function has not been clarified, the findings of this study demonstrate that Tej plays a crucial role in recruiting RNA helicases Vas and Spn-E to nuage through distinct domains, namely, Lotus and SRS. The results provide new insights into the regulation of stepwise piRNA precursor processing by Tej, Spn-E, and Vas in the initial phase of piRNA biogenesis prior to the ping-pong amplification cycle. Tej recruits these helicases for the engagement of the precursors involved in further processing of nuage, thereby also controlling the dynamics of these nuage components (Lin, 2023).

The results confirmed that the Tej Lotus domain recruited Vas to nuage, which is consistent with the fact that it enables Vas to hydrolyze ATP for RNA release (Jeske, 2017). This study newly identified that the SRS motif in Tej is responsible for Spn-E recruitment to nuage. Full deletion or single amino acid substitution of SRS significantly disrupted Spn-E recruitment to Tej granules in S2 cells, whereas further deletions of eight amino acids other than SRS, eSRS, were critical for recruiting Spn-E to nuage in the ovaries. This result raises a possibility that Tej, as well as other factors, may assist the recruitment of Spn-E to nuage in the ovaries. Another protein known as Tap, which is a fly counterpart of TDRD7 and harbors Lotus and eTudor domains, has previously been reported to participate in the piRNA pathway and interact with Vas (Jeske, 2017; Patil, 2014). However, since Tap lacks the SRS found in Tej, it is unlikely to be involved in the recruitment of Spn-E. The mouse homolog of Spn-E (TDRD9) is localized in both nuage and the nucleus in prespermatogonia, and might perform different functions that remain elusive. This finding suggests a possibility that the intrinsically nuclear protein Spn-E was deliberately recruited to nuage via Tej to exert a unique function, such as piRNA precursor processing. In contrast, the eTudor domain mainly contributes to Tej aggregation, which is consistent with previous studies showing that the eTudor domain is engaged in granulation by binding to its ligand sDMA (Lin, 2023).

Despite the unusual nuage granules of Tej-ΔeTudor, it mildly suppressed transposon expression. Notably, Tej-ΔeTudor displays interaction with Vas and Spn-E, albeit to a lesser extent, especially with Spn-E. The CL-IP results also supported these interactions as reported in S2 cells (Patil, 2010). Alternatively, Tej-ΔeTudor possibly may facilitate the association of other components with nuage activity for piRNA processing. Unlike the mutation of precursor transporter, nxf3, and the ping-pong cycle assistant, krimp, tej, as well as spn-E and vas mutants, exhibited the accumulation of piRNA precursors in the perinuclear region and a collapse of the ping-pong amplification. These results suggest that they function upstream during ping-pong amplification. Stalling of piRNA precursors was also observed when the recruitment of Vas or Spn-E to nuage was abolished by the loss of the Lotus or eSRS domains, respectively. Precursor accumulation was concentrated in the malfunctioning nuage or perinuclear region, which would result in a failure in precursor processing and cause TE upregulation (Lin, 2023).

Genetic analysis of nuage organization revealed that Spn-E and Tej occupy a higher hierarchical position than Vas at an earlier stage, which is inconsistent with a previous observation (Patil, 2010), possibly due to the fluctuation of nuage assembly and/or structure at a later stage in the mutants. In contrast, Tej and Spn-E are mutually dependent for the proper assembly of nuage granules because Spn-E is required for the proper localization of Tej within nuage. Moreover, Tej may form a relatively stable scaffold with Spn-E for nuage assembly, while a mobile fraction of Tej may contain Vas. These results suggest that Tej may facilitate the compartmentalization of Vas and Spn-E, as shown in CL-IP experiments and also reported in Bombyx germ cells, while the possibility cannot be excluded of simultaneous binding among these proteins. Further results with S2 revealed that the weak hydrophobic interaction between the proteins may contribute to the formation and regulation of membraneless structures on nuage. DEAD-box RNA helicase family members, including Vas homolog, reportedly form non-membranous, phase-separated organelles in both prokaryotes and eukaryotes, and the large IDR at the N-terminal region facilitates their aggregation by LLPS. In addition, the loss of IDR in Tej significantly suppressed the mobility of Tej and Vas; nevertheless, the TE repression was only mildly attenuated. Thus, Tej-ΔIDR may remain colocalized with Vas and Spn-E, facilitating the processing of piRNAs. Alternatively, the reduction of Vas mobility by the loss of Tej IDR could be compensated by other components in nuage. Only the localization of Vas was remarkably changed upon 1,6-hexanediol (1,6-HD) treatment in S2 cells, further supporting the finding that weak hydrophobic interaction controlled the dynamics of Vas, although a possibility of the unexpected effects by the 1,6-HD treatment cannot be excluded. It also cannot be excluded that 1,6-HD treatment might have impaired kinase and/or phosphatase activity. Hence, localization might have been affected by the changes in their phosphorylation status. The behavior of these proteins is seemingly influenced by their respective binding modes and properties with Tej. The interaction of Vas with Tej is affected by 1,6-HD and IDR region of Tej through the hydrophobic association, whereas that of Spn-E with Tej is more rigid, possibly contributing to the formation of the scaffold of nuage. In conclusion, Tej utilizes the eTudor domain for granule formation, whereas the IDR of Tej appears to maintain the assemble of Tej granules, controlling the mobility of Vas in nuage (Lin, 2023).

Membraneless macromolecular nuage contains more than a dozen components, including Vas and Tej that harbor IDRs, which could contribute to the dynamics of nuage and impact the efficient production of piRNAs. Nuage also contains piRNA precursors and TE RNAs that are processed therein; their unique or specific propensities may affect nuage assembly and function. Further investigation of those proteins and RNA components will shed light on the regulatory mechanisms underlying the formation and dynamics of nuage to promote each sequential step of piRNA biogenesis (Lin, 2023).

The LOTUS domain is a conserved DEAD-box RNA helicase regulator essential for the recruitment of Vasa to the germ plasm and nuage

DEAD-box RNA helicases play important roles in a wide range of metabolic processes. Regulatory proteins can stimulate or block the activity of DEAD-box helicases. This study shows that LOTUS (Limkain, Oskar, and Tudor containing proteins 5 and 7) domains present in the germline proteins Oskar, TDRD5 (Tudor domain-containing 5; Tejas), and TDRD7 (Tapas) bind and stimulate the germline-specific DEAD-box RNA helicase Vasa. Crystal structure of the LOTUS domain of Oskar in complex with the C-terminal RecA-like domain of Vasa reveals that the LOTUS domain occupies a surface on a DEAD-box helicase not implicated previously in the regulation of the enzyme's activity. It was shown that, in vivo, the localization of Drosophila Vasa to the nuage and germ plasm depends on its interaction with LOTUS domain proteins. The binding and stimulation of Vasa DEAD-box helicases by LOTUS domains are widely conserved (Jeske, 2017).

This study provides molecular insight into the function of animal LOTUS domain proteins, factors involved in diverse germline functions. The DEAD-box helicase Vasa interacts with the LOTUS domains of Oskar, TDRD5/Tejas, and TDRD7/Tapas but not with MARF1. In Drosophila, interaction with LOTUS domain proteins is required for Vasa localization to the nuage and germ plasm. Structural and functional analyses of the LOTUS-Vasa interaction uncovered a key role of a C-terminal extension present in only a subset of LOTUS domains, pointing to two LOTUS domain subclasses with distinct functions in animals. The eLOTUS domain of Oskar, TDRD5, and TDRD7 not only interacts with Vasa but also stimulates its helicase activity. The mLOTUS domains present in MARF1 lack this extension and very likely have a distinct role within the germline that will need to be addressed in the future. While Drosophila TDRD5 (Tejas) and TDRD7 (Tapas) contain a single eLOTUS domain, some TDRD5 and TDRD7 proteins from other animals harbor mLOTUS domains in addition to their N-terminal eLOTUS domain. Whether the mLOTUS domains from MARF1, TDRD5, and TDRD7 have related activities or are functionally distinct remains to be determined (Jeske, 2017).

The Drosophila eLOTUS domain proteins Oskar, Tejas, and Tapas have been considered to be scaffolding proteins whose function is to recruit Vasa and other germline factors to germ plasm or the nuage. While LOTUS domains were originally predicted to be RNA-binding domains, attempts to detect any RNA-binding activity of the eLOTUS domain of Oskar have failed. The present study uncovered a conserved function of eLOTUS domains in binding and stimulating a DEAD-box RNA helicase, thus attributing an active regulatory role to Oskar, Tejas, and Tapas in the germline. The stimulation of the ATPase activity of Vasa by the eLOTUS domain seems universal, but its consequence and function within the germline are unknown. In Drosophila, Vasa stimulation by Tejas and/or Tapas in the nuage might be involved in the piRNA pathway, whereas Vasa stimulation by Oskar in the pole plasm likely has a distinct role. Vasa was suggested to activate translation of mRNAs in the egg chamber through recruitment of eIF5B, which catalyzes ribosomal subunit joining to form elongation-competent ribosomes. Vasa has been shown to physically interact with eIF5B in yeast two-hybrid assays and pull-down experiments from lysates. A Vasa region that extends C-terminally from the helicase core was shown to be required for the eIF5B interaction, which raised the question of whether eLOTUS and eIF5B jointly or mutually exclusively bind to Vasa. Attempts were made to test this in GST pull-down assays with recombinant proteins. However, surprisingly, no interaction of Vasa with GST-eIF5B or any change in Vasa's ATPase activity in the presence of eIF5B was detected. It is concluded that Vasa and eIF5B do not physically interact and that the recruitment of eIF5B by Vasa might be mediated through RNA or other proteins. It is equally plausible that Vasa's role in translation might be that of a DEAD-box RNA helicase involved in remodeling RNA-protein complexes. Given its importance in germline biology, the mechanism by which Vasa promotes translation of mRNAs merits thorough re-examination (Jeske, 2017).

In the nuage, Vasa is essential for the secondary piRNA biogenesis pathway, also known as the Ping-Pong cycle. Bombyx Vasa associates with the Piwi proteins Siwi and Ago3, two major players in the Ping-Pong cycle in the germ plasm. Within the Ping-Pong cycle, Siwi is loaded with piRNAs, and the complex binds and cleaves transposon mRNAs in an orientation antisense to piRNAs. The cleavage products are then loaded into Ago3, and the complex recognizes and cleaves piRNA cluster transcripts, leading to specific amplification of piRNAs that target transposon mRNAs present in the cell. Vasa is required for the safe handover of transposon mRNA fragments from Siwi to Ago3. Furthermore, the ATPase activity of Vasa is necessary for the release of transposon RNAs from Siwi-piRNA complexes after cleavage. It is therefore possible that stimulation of Vasa by the Tejas and/or Tapas eLOTUS domains is required for high efficiency of the Ping-Pong cycle. The higher activity of Tejas compared with Tapas that was detected might be reflected in vivo by its dominant role in transposon silencing within the nuage (Jeske, 2017).

LOTUS domains are not restricted to animals but are also present in bacteria, fungi, and plants-organisms without a Vasa ortholog. From sequence alignments, it appears that bacterial, fungal, and plant LOTUS domains lack the particular C-terminal extension, and it will be interesting to investigate and compare their function with that of mLOTUS domains of animal proteins, such as MARF1 (Jeske, 2017).

Repression of retroelements in Drosophila germline via piRNA pathway by the Tudor domain protein Tejas

The Piwi-interacting RNAs (piRNAs) have been shown to safeguard the animal germline genome against deleterious retroelements. Many factors involved in the production of piRNAs localize to nuage, a unique perinuclear structure in animal germline cells, suggesting that nuage may function as a site for processing of germline piRNAs. This study reports a conserved yet uncharacterized component of the germline piRNA pathway, Tejas (Tej), which localizes to nuage. tej is required for the repression of some retroelements and for the production of sufficient germline piRNAs. The localization of Tej to nuage depends on vasa (vas) and spindle-E (spn-E) while it regulates the localization of Spn-E, Aubergine (Aub), Argonaute3 (Ago3), Krimper (Krimp), and Maelstrom (Mael) to nuage. Aub, Vas, and Spn-E physically interact with Tej through the N terminus containing the conserved Tejas domain, which is necessary and sufficient for its germline function. Aub and Spn-E also bind to the tudor domain at the C terminus. These data suggest that Tej contributes to the formation of a macromolecular complex at perinuclear region and engages it in the production of germline piRNAs (Patil, 2010).

This study reports that a new member of the germline piRNA pathway, tej, is required for the production of sufficient germline piRNAs to repress retroelements in Drosophila. tej encodes a tudor domain protein localized to nuage, a potential processing site of germline piRNAs. tej regulates the localization of other piRNA components, including the piRISC components Aub and Ago3, to nuage. Further, Tej physically interacts with Vas, Spn-E, and Aub, and the interaction with Aub is independent of sDMA. Together with previous observations, these genetic and physical interaction studies suggest that piRNA components may form a macromolecular complex at the perinuclear region by being engaged in processing retroelement RNA into piRNAs in the ping-pong cycle. The hierarchical interaction of genes coding for components of the nuage, with regard to localization of the proteins they encode, may indicate that these proteins act sequentially in the ping-pong cycle. Vas may first become loaded onto nuage. Then Tej may act together with Vas and Spn-E to unwind retroelement transcripts and engage Aub in processing them into germline piRNAs. Unlike others, tej is an unusual piRNA component mutant that does not show polarity defects. This could be due to piRNA-independent functions of other nuage components in the polarity formation, which does not involve tej. Alternatively, the establishment of polarity may depend on one or more specific piRNAs that require other piRNA or nuage components, but not tej function, to be generated. Profiling of piRNA in tej mutant will provide some insights to address this issue (Patil, 2010).

Search PubMed for articles about Drosophila Tejas

Jeske, M., Muller, C. W., Ephrussi, A. (2017). The LOTUS domain is a conserved DEAD-box RNA helicase regulator essential for the recruitment of Vasa to the germ plasm and nuage. Genes Dev. 31(9):939-952. PubMed ID: 28536148

Lin, Y., Suyama, R., Kawaguchi, S., Iki, T. and Kai, T. (2023). Tejas functions as a core component in nuage assembly and precursor processing in Drosophila piRNA biogenesis. J Cell Biol 222(10):e202303125. PubMed ID: 37555815

Patil V. S., Kai, T. (2010). Repression of retroelements in Drosophila germline via piRNA pathway by the Tudor domain protein Tejas. Curr Biol. 20(8):724-730. PubMed ID: 20362446

Patil V. S., Anand, A., Chakrabarti, A., Kai, T. (2014). The Tudor domain protein Tapas, a homolog of the vertebrate Tdrd7, functions in the piRNA pathway to regulate retrotransposons in germline of Drosophila melanogaster. BMC Biol. 12:61. PubMed ID: 25287931

Yabuta Y., Ohta, H., Abe, T., Kurimoto, K., Chuma, S., Saitou, M. (2011). TDRD5 is required for retrotransposon silencing, chromatoid body assembly, and spermiogenesis in mice. J Cell Biol. 192(5):781-795. PubMed ID: 21383078

date revised: 5 December, 2023

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