Studies on Mono-Uridylation of Pre-MicroRNAs

Abstract

MicroRNAs (miRNAs) are single-stranded RNAs of ~22 nucleotide (nt) in length that function as guide molecules of gene silencing. Biogenesis of miRNA involves multiple maturation steps. RNase III Drosha initiates miRNA maturation by cleaving a primary miRNA (pri-miRNA) transcript and releasing a hairpin shaped precursor miRNA (pre-miRNA) with a 2-nt 3′ overhang. The downstream maturation factor, Dicer, recognizes the 2-nt 3′ overhang structure to selectively process pre-miRNAs. Biogenesis of the let-7 miRNA family, one of the most ancient and highly conserved miRNAs, is suppressed in embryonic stage and in certain cancer cells. It was previously reported that Terminal uridylyl transferase 4 (TUT4) down-regulates let-7 miRNA biogenesis by oligo-uridylating let-7 precursor (pre-let-7) in mouse embryonic stem cells and that a pluripotency marker Lin28 acts as a processivity factor of TUT4. Here I find that TUTs positively controls let-7 biogenesis via mono-uridylation of pre-let-7 in a Lin28-independent manner. I identify TUT7/ZCCHC6, TUT4/ZCCHC11, and TUT2/PAPD4/GLD2 as the terminal uridylyl transferases responsible for pre-miRNA mono-uridylation. Such mono-uridylation enhances Dicer processing by generating an optimal 3′ 2nt overhnag end structure of pre-let-7 for Dicer recognition. I further suggest that miRNAs is subdivided into two groups, group I (which has 2nt 3′ overhang) and group II (which has 1nt 3′ overhang) based on the end structure of the precursor and dependency for mono-uridylation. Unlike prototypic pre-miRNAs (group I), group II pre-miRNAs acquire a shorter (1-nt) 3′ overhang from Drosha processing and therefore require a 3′-end mono-uridylation for Dicer processing. The majority of let-7 miRNAs and miR-105 belong to group II. The TUTs act specifically on dsRNAs with a 1-nt 3′ overhang, thereby creating a 2-nt 3′ overhang for optimal Dicer processing. Depletion of TUTs reduces let-7 levels and disrupts let-7 function in cells. While the let-7 suppressor, Lin28, induces inhibitory oligo-uridylation in embryonic stem cells, mono-uridylation occurs in somatic cells lacking Lin28 to promote let-7 biogenesis. My study reveals functional duality of uridylation and introduces TUT7/4/2 as novel components of the miRNA biogenesis pathway.