Identification and characterization of the 21U RNA processing enzyme in Caenorhabditis elegans
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Abstract
The piRNA pathway is a widely-represented, small RNA-based mechanism that takes part in transposon silencing. It is mostly active in the germline and early embryos. Even though the piRNA pathway is present in many species, mechanistic details of the pathway vary significantly. For instance, in many animals piRNAs are produced from long precursor transcripts that are processed afterward to form multiple mature piRNAs, whereas in C. elegans piRNA precursors are transcribed from individual loci that each produce a single piRNA. PiRNAs in C. elegans are called 21U RNA, because they are 21 nucleotide long and have strong 5’ U bias. 21U RNA precursors are roughly 28 nt long and contain a 5’ cap. To become a 21U RNAs, these precursors undergo maturation both at the 5’ and 3’ end.
The mechanism of the 5’ end processing of 21U RNA precursors in C. elegans is yet unknown. Our lab previously identified a protein complex, named PETISCO, that binds 21U RNA precursors and is required for their processing. Interestingly, PETISCO has two mutually exclusive interactors: PID-1 and TOST-1. PID-1-PETISCO only plays a role in 21U RNA maturation, while TOST-1-PETISCO has a different and still unknown maternal essential function, but does not affect 21U RNA biogenesis. However, how PETISCO can help 21U RNA production is still unclear.
I started with the purification of PETISCO proteins and obtained working antibodies against TOFU-6, IFE-3, and PID-3. In collaboration with Sebastien Falk (Vienna), the crystal structure of PETISCO was obtained and the effects of the disrupting interactions within PETISCO were tested in vivo.
In my second project, I identified a novel nuclease complex, which had been named PUCH (Precursor of 21U 5’ end Cleavage Holoenzyme), with an essential role in piRNA precursor processing. PUCH is characterized by three subunits, TOFU-1, TOFU-2, and one of the newly identified 21U RNA pathway factors SLFL-3 or SLFL-4. The active center of PUCH is formed by Schlafen-like domains, present in all four proteins. I proved the activity of PUCH in vivo and in vitro. Analysis of the substrate requirements showed that PUCH needs m7G-cap and U in position three to perform the cleavage.
Again, in collaboration with Sebastian Falk, the link between PETISCO and PUCH was identified. Interaction between TOFU-6 and TOFU-1 was demonstrated in vitro and crystal
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structure of this interaction was obtained. CRISPR/CAS9 genome editing we disrupted the interaction between PUCH and PETISCO and confirmed the relevance of this interaction for 21U RNA maturation in vivo.
Thus, I identify PUCH as a novel 5’ end processing complex that drives piRNA precursor maturation in C. elegans, in conjunction with the precursor-binding complex PETISCO. The catalytic center of PUCH consists of three different SLFN domains. Given the multitude of non-characterized SLFN homologs in mammalian genomes, PUCH may define a novel type of multi-subunit nuclease enzymes with many different yet unknown functions.