Josh I Steinberg (1,2), Wolfram Gruhn (3), Daisy Rubio (1,) Azim Surani (3), Andrea J Schorn (1)
1 Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA; 2 Stony Brook University Medical Scientist Training Program, Stony Brook, NY 11794, USA; 3 The Gurdon Institute, University of Cambridge, UK

Endogenous retroviruses (ERVs) utilize host tRNA as a primer for reverse transcription and replication, a hallmark of long terminal repeat (LTR) retroelements. Their dependency on tRNA makes these elements vulnerable to targeting by small RNAs derived from the 3′-end of mature tRNAs (3′-tRFs), which are highly expressed during epigenetic reprogramming and potentially protect many tissues in eukaryotes. We found high levels of 3'-tRF in male and female mouse primordial germ cells coincide with elevated ERV burden at sex-specific time points. tRFs are expressed when heterochromatin levels decline, before the onset of piRNA production in males and in the absence of piRNAs in females.

3'-tRFs inhibit ERV activity by blocking reverse transcription but also by post-transcriptional silencing. Due to the perfect sequence complementarity of 3'-tRFs to endogenous retroviral sequences, they have tens of thousands of targets in mammalian genomes. We conducted a massively parallel reporter assay to determine target site rules for 3'-tRFs within the tRNA primer binding site of ERVs. Moreover, we found 3'-tRFs are licensed for bona fide RNA silencing by post-transcriptional modifications akin to piRNAs and small RNA classes in other organisms that have perfect sequence complementarity to transposon targets but evade target-directed degradation.