The protein protectors of fertility
|Journal||J. Cell Biol. 222(10):e202303125 (2023)|
|Title||Tejas functions as a core component in nuage and precursor processing in Drosophila piRNA biogenesis|
|Laboratory||Germline Biology Group〈Prof. KAI Toshie〉|
You're likely familiar with RNA, the molecule that plays an important role in protein production and gene expression control. Perhaps you're less familiar, however, with PIWI-interacting RNA (piRNA), a special type of RNA that protects the genome from mutations. Now, researchers in Japan have shed light on how these critical molecules are formed by the dynamics of several associated proteins in the germline of the fruit fly, Drosophila melanogaster.
In a new study published in the Journal of Cell Biology, researchers from Osaka University have clarified how the proteins Tejas (Tej), Vasa (Vas), and Spindle-E (Spn-E) contribute to the creation of piRNAs, which are found in the gonads and help protect the genome from mutations or deletions caused by transposons that may be passed on through reproductive cells.
In Drosophila, piRNAs first appear as long sequences, known as precursor transcripts, which are processed in membrane-less cellular compartments called ‘nuage' and turned into their shorter forms as functional piRNAs. These nuage are composed by the interactions of RNA-processing proteins, PIWI family proteins, and a group of proteins known as the Tudor-domain-containing (Tdrd) proteins. Of those, Tej, a Tdrd protein, has been previously shown to play a key role in the piRNA pathway, with a major reduction in piRNAs observed in the absence of Tej. However, the role that Tej plays in the generation of piRNA is not fully understood. The research team set out to investigate the molecular function of Tej during piRNA biogenesis.
"We first evaluated the detailed function of Tej in the Drosophila ovary and confirmed that Tej is involved in processing precursor transcripts with two RNA-processing helicases: Vas and Spn-E in the nuage," explains lead author of the study Lin Yuxuan. "In the mutant germ cells that lack Tej, we observed that Vas and Spn-E are not properly assembled in the nuage, indicating that Tej plays a critical role in recruiting Vas and Spn-E to the nuage."
By generating variants of Tej that lack specific regions of the protein, the investigators could identify which areas of Tej are crucial for interacting with Spn-E and modulating the mobility of Vas. They found that one distinct region, termed the 'Spn-E recruit site', contributes to the recruitment and maintenance of Spn-E in the nuage. Another distinct region, known as the intrinsically disordered region, affects both the ability of Vas to move within the nuage and the dynamics of other components of the nuage.
"Our study demonstrates that Tej is a key component of nuage formation and piRNA processing," says senior author Kai Toshie. "By recruiting Vas and Spn-E, Tej facilitates nuage formation, allowing the processing of piRNA precursor transcripts into their mature, functional forms."
Disruption of piRNA formation is known to cause infertility. The research team's insight into the formation of nuage and piRNA processing mediated by Tej in Drosophila germline may help to uncover the mechanisms underlying reproductive disorders, including infertility, and may contribute to the development of new fertility drugs.
PIWI-interacting RNAs (piRNAs) which protect genome from the attack by transposons, are produced and amplified in membrane-less 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. Here, we conduct a detailed analysis of the subcellular localization of fluorescently-tagged nuage proteins and behavior of piRNA precursors. Our 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. Our study also reveals that the low-complexity region of Tej regulates the mobility of Vas. Based on these results, we propose that Tej plays a pivotal role in piRNA precursor processing by assembling Vas and Spn-E into nuage and modulating mobility of nuage components.
piRNA biogenesis pathway in the Drosophila germline.
The Drosophila ovary has 16–20 ovarioles containing 14 different developmental stages of egg chambers. The piRNA precursors transcribed in the nucleus are transported to the nuage, which is located on the cytoplasmic side of the nuclear membrane. At the nuage, Tej, Vas, Spn-E, Krinmp, and other proteins accumulate, and Aub and Ago3 cleave piRNA precursors and complementary-sequenced transposons. In the nuage, Tudor domain proteins cooperate with PIWI family proteins, and piRNAs are generated via the ping-pong amplification pathway, which is conjugated with the degradation of transposon transcripts.
AlphaFold prediction of the interaction regions for Tej–Spn-E proteins.
Heterodimeric structures of Tej (right, red) and Spn-E (left, blue) are predicted using AlphaFold v2.2. The C-terminal conserved region of Spn-E (blue) predicted to interact with Tej-eSRS (red) was experimentally proven in the Drosophila ovary.
Tej interacts with Spn-E and Vas and has a role in processing piRNA precursors.
In Drosophila germ cells, Spn-E localizes to the nuage in the perinuclear membrane region via the eSRS motif in Tej. In contrast, the Lotus domain of Tej facilitates Vas localization to the nuage. This complex is involved in the processing of piRNA precursors, and the IDR of Tej provides mobility to the Tej–Vas complex in the nuage, contributing to its role as a component of a non-membrane organelle.