Comparative analysis of nematode small-RNA pathways using gametocyte specific factor 1 (GTSF-1)

Abstract

Small-RNAs (sRNAs) regulate gene expression by binding to an Argonaute (Ago) protein and interfering with mRNA transcription or translation. Evolutionary arms race with transposable elements has greatly diversified the small-RNA pathways in individual species. This resonates in the Zn finger protein Gametocyte specific factor -1 (GTSF-1), which are essential for sRNA pathways in many species but show evolutionary plasticity by acting at different steps of the pathway in different species. In mice and insects, GTSF-1 is a PIWI Argonaute-associated protein crucial for piRNA-directed silencing. It plays a pivotal role by enhancing the endoribonuclease activity of catalytic PIWI Agos (Arif et al., 2022). However, in the nematode C. elegans, GTSF-1 associates with the RNA-Dependent RNA Polymerase (RdRP) RRF-3, diverging from the conventional PIWI association (Almeida et al., 2018). RRF-3 is essential for the biogenesis of 26G-RNAs (siRNAs), which regulate gene expression in the germline. The observation gains significance as several nematode lineages independently lost PIWI and the entire piRNA pathway, yet retained RRF-3-like RdRPs (Sarkies et al., 2015). To investigate the conservation of GTSF-1 function within nematodes, we conducted a comparative analysis of small-RNA pathways in three nematode species: C. elegans, C. briggsae and P. pacificus, with a focus on GTSF-1 and the 26G-RNA pathway. Employing various methodologies including CRISPR-Cas9 transgenics, Next Generation Sequencing, Immunoprecipitation coupled to Mass-spectrometry, and AlphaFold2 predictions, we elucidated the interactions and functions of GTSF-1 within these nematode species. Our findings reveal an interaction between GTSF-1 and RRF-3 in C. briggsae and P. pacificus, crucial for fertility and the regulation of 26G-RNAs. We identified sequence and structural differences in nematode GTSF-1 homologs potentially explaining the loss of PIWI binding. Utilizing AlphaFold2, we identified key domains and residues for GTSF-1 binding to RRF-3, validated through in-vitro and in-vivo assays. We also described conserved functions of the PETISCO complex in C. briggsae embryogenesis and 21U-RNA biogenesis. Notably, we uncover a curious link between the PETISCO and 26G-RNA pathway in C. briggsae, potentially mediated through GTSF-1. In summary, these results shed light on the conservation and functional significance of small RNA pathways in clade V nematodes. Moreover, our study suggests ancient conservation of this association within nematodes, highlighting the functional adaptation of nematode GTSF-1 proteins. Through this work, we provide a compelling explanation for the loss of the highly conserved piRNA pathway across nematode lineages.