TUT7 controls the fate of precursor microRNAs by using three different uridylation mechanisms

Abstract

Terminal uridylyl transferases (TUTs) function as integral regulators of microRNA (miRNA) biogenesis. Using biochemistry, single-molecule, and deep sequencing techniques, we here investigate the mechanism by which human TUT7 (also known as ZCCHC6) recognizes and uridylates precursor miRNAs (pre-miRNAs) in the absence of Lin28. We find that the overhang of a pre-miRNA is the key structural element that is recognized by TUT7 and its paralogues, TUT4 (ZCCHC11) and TUT2 (GLD2/PAPD4). For group II pre-miRNAs, which have a 1-nt 3′ overhang, TUT7 restores the canonical end structure (2-nt 3′ overhang) through mono-uridylation, thereby promoting miRNA biogenesis. For pre-miRNAs where the 3′ end is further recessed into the stem (as in 3′ trimmed pre-miRNAs), TUT7 generates an oligo-U tail that leads to degradation. In contrast to Lin28-stimulated oligo-uridylation, which is processive, a distributive mode is employed by TUT7 for both mono- and oligo-uridylation in the absence of Lin28. The overhang length dictates the frequency (but not duration) of the TUT7-RNA interaction, thus explaining how TUT7 differentiates pre-miRNA species with different overhangs. Our study reveals dual roles and mechanisms of uridylation in repair and removal of defective pre-miRNAs. Synopsis Uridylation of miRNA precursors can either stimulate processing or trigger RNA degradation. This study shows how RNA overhang structure and the mode of TUTase binding facilitate differential uridylation of specific precursor types. Terminal uridylyl transferases (TUTs) exert multiple roles in miRNA biogenesis by uridylating precursor miRNAs, thereby determining their fates. For group II pre-miRNAs, which carry a 1-nt 3′ overhang, TUT7/4/2 restore the canonical end structure through mono-uridylation, promoting miRNA biogenesis. For 3′ trimmed pre-miRNAs, TUT7/4 distributively generate an oligo-U tail that triggers degradation. TUT7 distinguishes pre-miRNA species at the binding step and shows distinct binding frequency to different RNA substrates. Uridylation of miRNA precursors can either stimulate processing or trigger RNA degradation. This study shows how RNA overhang structure and the mode of TUTase binding facilitate differential uridylation of specific precursor types. © 2015 The Authors. Published under the terms of the CC BY 4.0 license