The ambivalence of the role of transposons in eukaryotic genomes is fascinating: these small nucleic acids mobile elements are at the same time motor of evolution by generating new functionalities and source of deleterious mutations when they integrate into essential genes. In fact, an uncontrolled mobilization of these elements in the germ line makes the animals sterile. In this context, the maintenance of the integrity of eukaryotic genomes depends on an impressive number of molecular processes, among which the genesis of small RNAs acting as guardian of the genome by controlling the mobilization and integration of transposons in the germ line.
These small RNAs, called piRNAs because they interact with PIWI (PIWI interacting RNAs) proteins, are 24-30 nucleotide RNAs. Expressed exclusively in the germ line, they pair with RNA transcripts from transposons to cause their degradation and prevent their reintegration as DNA in the genome. The primary biogenesis of these RNAs transcripts is based on long single-stranded RNA precursors, themselves transcribed from clusters of transposons. However, molecular mechanisms that identify single-stranded RNA as a precursor for piRNA biogenesis are poorly understood while this identification is crucial for targeting the mobile elements in a specific manner among the set of cellular RNAs.
In a recent study carried out in collaboration with the University of Geneva, we used the Drosophila melanogaster fly to address the issue of piRNAs biogenesis. From transgenic lines of Drosophila flies, we demonstrate that the artificial anchoring of specific factors of piRNAs biogenesis to a long RNA transcript is sufficient to identify the latter as a precursor for the piRNAs biogenesis in the ovaries (Figure). This results in fragmentation of the "reporter" transcript into thousands of piRNAs. These small RNAs are then loaded into PIWI proteins and amplified.
Armi (Armitage) is a factor involved in the biogenesis of piRNA. In this experiment, the tagged HA-Armi protein, fused or not to the N sequence which recognizes the BoxB box, is expressed specifically in the Drosophila ovaries with a reporter gene which contains the coding sequences of the Luciferase and LacZ genes from either side of a BoxB box. RNAs associated with PIWI proteins are isolated by immuno-precipitation and analyzed by high throughput sequencing.
We observe the directional genesis (5’->3’) of piRNAs from the LacZ gene in the case of the Armi N-HA construct which is capable of binding to the reporter gene while the HA-Armi construction is not functional.