A nanobody targeting the LIN28:let-7 interaction fragment of TUT4 blocks uridylation of let-7

Significance Terminal uridylyltransferases are writers of poly(U) tails in diverse RNA uridylation pathways. In the let-7 pathway, the LIN28:pre-let-7:TUTase ribonucleoprotein complex regulates the processing and maturation of the let-7 microRNA, and plays an important role in both physiology and disease. The presence of redundant TUTase orthologs and the diverse pathways poses challenges to revealing the molecular mechanisms of TUTases. We developed a precise tool to identify nanobodies that inhibited the LIN28:pre-let-7:TUTase interaction. The nanobody Nb-S2A4 specifically interacted with the LIN28:let-7 interaction fragment in TUT4, which is critical for oligouridylation and monouridylation of let-7. Our data illustrate that the nanobody tool is effective in distinguishing functions among protein orthologs and diverse pathways.
The LIN28:pre-let-7:TUTase ternary complex regulates pluripotency and oncogenesis by controlling processing of the let-7 family of microRNAs. The complex oligouridylates the 3′ ends of pre-let-7 molecules, leading to their degradation via the DIS3L2 exonuclease. Previous studies suggest that components of this complex are potential therapeutic targets in malignancies that aberrantly express LIN28. In this study we developed a functional epitope selection approach to identify nanobody inhibitors of the LIN28:pre-let-7:TUT4 complex. We demonstrate that one of the identified nanobodies, Nb-S2A4, targets the 106-residue LIN28:let-7 interaction (LLI) fragment of TUT4. Nb-S2A4 can effectively inhibit oligouridylation and monouridylation of pre-let-7g in vitro. Expressing Nb-S2A4 allows maturation of the let-7 species in cells expressing LIN28, highlighting the therapeutic potential of targeting the LLI fragment.