Pseudouridine guides germline small RNA transport and epigenetic inheritance.
Epigenetic modifications that arise during plant and animal development, such as DNA and histone modification, are mostly reset during gamete formation, but some are inherited from the germline including those marking imprinted genes. Small RNAs guide these epigenetic modifications, and some are also inherited by the next generation. In C. elegans, these inherited small RNAs have poly (UG) tails, but how inherited small RNAs are distinguished in other animals and plants is unknown. Pseudouridine (?) is the most abundant RNA modification but has not been explored in small RNAs. Here, we developed novel assays to detect ? in short RNA sequences, demonstrating its presence in mouse and Arabidopsis microRNAs and their precursors.  We also detected substantial enrichment in germline small RNAs, namely epigenetically activated siRNAs (easiRNAs) in Arabidopsis pollen, and PIWI-interacting piRNAs in mouse testis. In pollen, pseudouridylated easiRNAs are produced by RNA polymerase IV and localized to sperm cells. We found that PAUSED/HEN5 (PSD), the plant homolog of Exportin-t, interacts genetically with ? synthase and is required for transport of easiRNAs into sperm cells from the vegetative nucleus.  We further show that PSD is required for the triploid block: chromosome dosage-dependent seed lethality that is epigenetically inherited from pollen. We propose that ? has a conserved role in marking inherited small RNAs in the germline. Overall design: Mouse testis small RNA sequencing after a) N-cyclohexyl-N'-ß-(4-methylmorpholinium) ethylcarbodiimide (CMC) treatment and b) pseudouridine immunoprecipitation.