Boseon Kim1,2,†, Minju Ha1,2,†, Luuk Loeff3,†, Hyeshik Chang1,2, Dhirendra K Simanshu4, Sisi Li4, Mohamed Fareh3, Dinshaw J Patel4, Chirlmin Joo3,* & V Narry Kim1,2,**
1 Center for RNA Resear ch, Institute for Basic Science, Seoul, Korea
2 School of Biological Sciences, Seoul National University, Seoul, Korea
3 Kavli Institute of NanoScience, Department of BioNanoScience, Delft University of Technology, Delft, The Netherlands
4 Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY, USA
*Corresponding author.
**Corresponding author.
†These authors contributed equally to this work
Abstract
Terminal uridylyl transferases (TUTs) function as integral regulators of microRNA (miRNA) biogenesis. Using biochemistry, singlemolecule, 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 30 overhang, TUT7 restores the canonical end structure (2-nt 30 overhang) through monouridylation, thereby promoting miRNA biogenesis. For pre-miRNAs where the 30 end is further recessed into the stem (as in 30 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.
Keywords : precursor microRNA; single-molecule fluorescence; TUT4 (ZCCHC11); TUT7 (ZCCHC6); uridylation