Non-coding RNA profiling by high throughput sequencing
Summary
PIWI-interacting RNAs (piRNAs) function in the nucleus and cytoplasm of animal germ cells to suppress mobile genetic elements. In the mouse male germline, biogenesis of MIWI2-bound nuclear piRNAs depends on endonuclease activity of cytosolic MILI, but the process is poorly understood. Here we use a mouse model expressing an artificial piRNA precursor to show that MILI slicing of the precursor generates a 16-nt by-product and a pre-piRNA intermediate that requires 3สน end processing to mature as a new piRNA. The ability to use the slicer products requires ATPase activity of the RNA helicase MVH, as the catalytic-dead Mvh mutant mice (Mvh-/KI) fail to convert the intermediate into piRNAs, abrogating biogenesis of MIWI2 piRNAs. This results in an early arrest in spermatogenesis and de-repression of transposons. Furthermore, the mutant MVH protein is dominant-negative (Mvh+/KI) as it causes a late-spermatogenic arrest by trapping complexes containing the mysterious pachytene piRNAs and slicer products, uncovering a role for the protein beyond the embryonic germline. In contrast, we find that the ATPase activity of TDRD9 is dispensable for piRNA biogenesis, but is essential for silencing by MIWI2. Our studies implicate distinct RNA helicases in specific steps along the mammalian nuclear piRNA pathway.
Overall design
Testicular piRNAs and longer RNA species associated with MVH, MILI, MIWI or MIWI2 were immunoprecipitated and sequenced. The effect of TDRD9 and MVH mutation on piRNA pathway was investigated.
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