The movement of DNA sequences within a genome threatens the execution of highly orchestrated molecular processes, making it especially dangerous within cells of the germline of an organism that transmit its genetic information. Nature has therefore evolved defence mechanisms that safeguard the genome by preventing the uncontrolled proliferation and movement of transposable elements (TEs). So-called PIWI proteins control transposon activity in the germline by binding to small RNA co-factors called piRNAs. As such, the biogenesis of these piRNAs, which regulate target specificity, is a crucial step in the inactivation of TEs. Initially transcribed as precursor RNAs, piRNAs depend on a series of coordinated post-transcriptional processing steps for their maturation. However, the enzymatic machineries that accomplish these tasks are diverse and many components are still missing.
Building on a previously published genome-wide RNA interference (RNAi) screens, and combining genetics with structural biology and biochemistry, the labs of René Ketting and Sebastian Falk have identified a novel nuclease in C. elegans that mediates sequence-specific cleavage of precursor piRNAs. Unlike the nucleases of mice and fruit flies, worms have evolved a nuclease comprising three Schlafen domain proteins, TOFU-1, TOFU-2 and one of either SLFL-3 or SLFL-4 paralogs. The authors demonstrate that nuclease activity depends on the association of all three proteins. Named PUCH for Precursor of 21U RNA 5’-end Cleavage Holoenzyme, the complex specifically recognizes 5’-capped piRNA precursors with a uracil at position 3. The authors conclude that this specificity likely explains the extreme bias for 5’ uracil in mature piRNAs. A single point mutation that abrogates PUCH activity is sufficient to almost entirely abrogate piRNA maturation.
Although the inactivation of TEs by piRNAs is conserved across all species, their biogenesis appears to be a case of convergent evolution. However, while different species have evolved their own piRNA biogenesis machineries, the processing and maturation of piRNAs takes place on the outer mitochondrial membrane in evolutionarily diverse species. “Why this reaction takes place on the surface of mitochondria is a big mystery”, says study co-leader Sebastian Falk. “What is clear, however, is that disrupting the localization of PUCH impairs piRNA maturation.” Further studies will be required to determine why the mitochondrial outer membrane is utilized for this apparently unrelated process.
Schlafen and Schlafen-like domains have been found in a variety of mammalian proteins implicated in innate immunity as well as in virulence factors of the monkeypox family of viruses. The composite nature of the PUCH nuclease involved in piRNA biogenesis raises intriguing questions about the potential role of Schlafen domain-containing proteins in innate immune defence mechanisms against foreign nucleic acids. “It’s conceivable that Schlafen proteins have been repurposed to engineer nucleases with unique specificities that protect cells from infectious nucleic acids”, says Sebastian Falk. Collaborator René Ketting suspects that Schlafen proteins may have a wider, conserved role in innate immunity. “Schlafen proteins may represent a previously unknown molecular link between immune responses in mammals and deeply conserved RNA-based mechanisms that control TEs.” Both Rene Ketting and Sebastian Falk agree that “the implications for innate immune biology are, therefore, quite profound.”
The work was a collaboration between a genetics lab and a structural biology lab in an example of inter-disciplinary science. “It’s amazing how different people can look at the same thing with completely different eyes,” says Sebastian Falk. “Without this collaboration, the discovery of PUCH and its role in piRNA biogenesis would not have been possible.”
Podvalnaya N, Bronkhorst AW, Lichtenberger R, Hellmann S, Nischwitz E, Falk T, Karaulanov E, Butter F, Falk S, Ketting RF: piRNA processing by a trimeric Schlafen-domain nuclease.Nature. 2023