Abstract
MicroRNAs (miRNAs) associated with Argonaute proteins (AGOs) regulate gene expression in mammals. miRNA 3’ ends are subject to frequent sequence modifications, which have been proposed to affect miRNA stability. However, the underlying mechanism is not well understood. Here, by genetic and biochemical studies as well as deep sequencing analyses, we find that AGO mutations disrupting miRNA 3’ binding are sufficient to trigger extensive miRNA 3’ modifications in HEK293T cells and in cancer patients. Comparing these modifications in TUT4, TUT7 and DIS3L2 knockout cells, we find that TUT7 is more robust than TUT4 in oligouridylating mature miRNAs, which in turn leads to their degradation by the DIS3L2 exonuclease. Our findings indicate a decay machinery removing AGO-associated miRNAs with an exposed 3’ end. A set of endogenous miRNAs including miR-7, miR-222 and miR-769 are targeted by this machinery presumably due to target-directed miRNA degradation.
Highlights
MicroRNAs associated with Argonaute proteins (AGOs) regulate gene expression in mammals. miRNA 3’ ends are subject to frequent sequence modifications, which have been proposed to affect miRNA stability
MicroRNAs are a class of small non-coding RNAs that serve as master regulators of gene expression in eukaryotic cells1. miRNAs bind to Argonaute proteins (AGO), with their 5′ end embedded inside the AGO MID domain and the 3′ end docked at the AGO PAZ domain[2]
A set of terminal nucleotidyltransferases (TENTs), including TUT4 (ZCCHC11/TENT3A/PAPD3) and TUT7 (ZCCHC6/TENT3B/ PAPD6) are responsible for adding non-templated nucleotides to the 3′ ends of miRNAs, whereas 3′–5′ exonucleases shorten the miRNAs by removing nucleotides from the 3′ end20–22. 3′ uridylated or adenylated isomiRs have different stabilities compared to the cognate miRNAs23–25 and plant miRNAs uridylated by HESO1 and URT1 at the 3′ termini are removed by 3′–5′ exonucleases[26,27]
Summary
MicroRNAs (miRNAs) associated with Argonaute proteins (AGOs) regulate gene expression in mammals. miRNA 3’ ends are subject to frequent sequence modifications, which have been proposed to affect miRNA stability. Tudor-SN cleaves AGO-bound miRNAs containing CA and UA dinucleotides, playing an important role in regulating cell cycle transition[13] Another mechanism to achieve miRNAspecific decay is target-directed miRNA degradation (TDMD), in which extensively paired targets induce miRNA turnover[14]. We investigate how 3′ modifications lead to miRNA decay by generating mutations in the AGO PAZ domain These mutations are sufficient to trigger extensive miRNA 3′ end modifications in cultured cells and in vivo, suggesting a machinery monitoring the status of the miRNA 3′ end. We find that these AGO-bound miRNAs with exposed 3′ ends are oligouridylated by both TUT4 and TUT7 and subsequently degraded by DIS3L2. We provide evidence that a set of endogenous miRNAs, including miR-7, miR-222, and miR769, which are likely under regulation of TDMD, are targeted by the TUT-DIS3L2 machinery in HEK293T cells
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