title: Dissecting the Mechanism of piRNA Biogenesis and Function in Adult Somatic Stem Cells of the Murine Hippocampal Niche
Creators: name: Tuntevski, Kiril
Publisher: name: The Open University
Year: 2025

description: PIWI-Interacting RNAs (piRNAs) are small regulatory non-coding RNAs with a pivotal role in the maintenance of germline stem cell pools and male fertility by epigenetic regulation and post-transcriptional silencing of transposable elements (TEs) and other transcripts. Recently, we demonstrated that piRNAs, in concert with PIWIL2, are required for the maintenance of proper adult neural stem cell (aNSCs) fate in the mouse hippocampus; however, mammalian piRNA biogenesis is elusive, and the licencing mechanism for piRNA biogenesis is unknown in aNSCs. To identify the <i>bona fide</i> piRNA genomic cluster loci we investigated small non-coding RNAs derived from primary mouse aNSCs. Analyses indicate that these loci share no common sequence motifs. In contrast, were able to identify RNA secondary structures shared between different piRNA precursors, as well as potential RNA binding proteins (RBPs) motifs immediately adjacent to these secondary structures. We posit that these structures flag piRNA precursors for their processing by the PIWI-RISC complexes in concert with additional RBPs. 
Concomitantly, the expression dynamics of known piRNA biogenesis proteins, such as MOV10, TDRKH, and interestingly TDP43, are consistent with the maturation dynamics of piRNAs observed in aNSCs, suggesting the possible implication of these RBPs to the piRNA-biogenesis in this cell model. To address this question, we focused on the top represented <i>bona fide</i> piRNA precursor(s), and found it mapping a 5’ tRNA fragment (5’ tRF tRNA-Gly-GGY piRNA locus). These 5’ tRFs and many others are associated with PIWIL2, MOV10 and TDP43 by cross-linking and co-immunoprecipitation and RNA sequencing (CLIP-Seq). Moreover, we also show that these sequences are resistant to acid-base RNA endonuclease T1 digestion, suggesting their 2’-O-methylation status. 
<i>In silico</i> simulations predict the secondary structures in the 5’ segment of the identified tRNA precursors is guanosine-quadruplex (GQ). To test whether this structure is involved in the process of piRNA biogenesis we incubated cells with pyridostatin, a drug known for GQ stabilisation, and found that this treatment impairs the maturation of this tRFs-derived piRNAs. Consistently, Piwil2-CLIP associated sequences lost their resistance to RNAse T1 treatment, but increased their affinity to complexes associated with GQ-binding MOV10 and TDP43 proteins, where they maintain RNAse T1 resistance. 
To evaluate the biological relevance of the tRF-derived piRNA we sought to sequester tRF-loaded-PIWIL2 functioning through an antisense oligonucleotide (antago-piR). We found that this antago-piR was able to co-localise to PIWIL2-dense regions of the perinuclear cytoplasm. Furthermore, we find that treatment with the antago-piR prevents cytotoxicity induced by differentiation of wild-type (WT) aNSCs, and doublecortin-positive cells failed to exit the cell cycle, suggesting that tRNA-Gly-GGY is required for the maintenance of both quiescent, as well as pro-neurally differentiating cells. Together, these findings elucidate the role of GQs and RBPs, such as MOV10 and TDP43, in the mechanism of piRNA biogenesis in aNPCs, and implicate the maturation of tRFs-derived piRNAs in the regulation of cell viability and differentiation.