HENMT1 restricts endogenous retrovirus activity by methylation of 3'-tRNA fragments

Long terminal repeat (LTR) retroelements such as endogenous retroviruses (ERVs) utilize host tRNA as a primer for reverse transcription, and are thus susceptible to silencing by small RNAs derived from the 3'-end of mature tRNAs (3'-tRFs). Rigorous quantification reveals that 3-tRF amounts are not directly proportional to tRNA levels, instead, 3-tRFs of specific isodecoder tRNAs are highly enriched in a pattern conserved between mouse and human. We found that 3-tRFs are 2-O methylated by the small RNA methyltransferase HENMT1 protecting them from degradation and promoting ERV silencing. In the absence of HENMT1, 3-tRFs are subjected to non-templated tailing by the terminal nucleotidyltransferases TUT4 and TENT2 that regulate small RNA turnover. Due to the perfect sequence complementarity of 3-tRFs to endogenous retroviral sequences, they have thousands of targets in mammalian genomes. We conducted a massively parallel reporter assay using Mus musculus particle type D, a highly active murine ERV, to determine target site rules for 3-tRFs. Our results suggest that HENMT1 not only stabilizes germline integrity but also serves transposon control by 3-tRFs in the soma.