TENT2, TUT4 AND TUT7 SELECTIVELY REGULATE MIRNA SEQUENCE AND ABUNDANCE
Terminal nucleotidyl transferases (TENTs) generate miRNA isoforms (isomiRs) by posttranscriptional 3' sequence modifications (tailing). These modifications are subject to temporal or spatial regulation in development and diseases, including cancer, suggesting they may play a functional role. While it is well-established that uridylation of the miRNA let-7 precursor has a profound impact on its biogenesis, little is known as to how the tailing of other miRNAs affects their function and level in mammals. Here, to systematically investigate how TENTs regulate miRNA sequence and abundance, we generated a set of human HEK293T cell lines in which the major tailing enzymes: TENT2, TUT4, and TUT7, were knocked out individually or in combination. Deep sequencing analyses confirmed that uridylation or adenylation of miRNAs decreased accordingly and could be rescued by ectopic expression of the corresponding tailing enzyme(s). Interestingly, a significant reduction of 3' modifications was only observed when all three TENTs were depleted, indicating TUT4/7-mediated uridylation and TENT2-mediated adenylation compensate for each other. Individually, TENT2 contributes to guanylation and uridylation in addition to adenylation on mature miRNAs. TUT4 uridylates most miRNAs but prefers miRNA substrates ending with a purine. Despite being expressed and having a higher activity in vitro, TUT7 is dispensable for most uridylated isomiRs observed in cells. While TENT2 specifically modulates miRNA sequences, removing TENT2 has only a marginal impact on miRNA levels in HEK293T cells, arguing that TENT2 may not play a significant role in regulating miRNA stability. In contrast, abolishing uridylation led to the upregulation of a set of miRNAs. We identified miR-138 and miR-222, among others, that are regulated by TUT4/7-mediated uridylation via distinct mechanisms. Our results uncovered the molecular basis for selective but coordinated actions of TENTs, highlighted the precise control of different 3' miRNA modifications in cells, and paved the way to investigate their functional impact in cancer.