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.
Highlights
MicroRNAs are generated by multiple maturation steps that consist of two endonucleolytic reactions (Ha & Kim, 2014)
Terminal uridylyl transferases (TUTs) share a common catalytic motif consisting of a nucleotidyl transferase (Ntr) and the poly(A) polymerase-associated (PAP) domain (Kwak & Wickens, 2007; Martin & Keller, 2007)
Given that the recombinant protein was produced in E. coli and purified to homogeneity, our result indicates that TUT7 does not require any additional cofactors for pre-miRNA monouridylation (Fig 1B and D)
Summary
MicroRNAs (miRNAs) are generated by multiple maturation steps that consist of two endonucleolytic reactions (Ha & Kim, 2014). Ago contributes to the production of truncated species by cleaving pre-miRNAs in the middle of the 30 strand. There is little evidence that ac-pre-miRNAs generate mature miRNAs with an exception of ac-pre-miR-451 that is shorter than others and trimmed further into mature miRNA (Cheloufi et al, 2010; Cifuentes et al, 2010; Yang et al, 2010; Yoda et al, 2013). It remains unclear whether acpre-miRNAs have a certain biological role in general or whether they are mostly degradation intermediates. The molecular mechanism of how the truncated pre-miRNAs are removed is largely unknown
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