Mammalian DIS3L2 exoribonuclease targets the uridylated precursors of let-7 miRNAs - PubMed (original) (raw)

Mammalian DIS3L2 exoribonuclease targets the uridylated precursors of let-7 miRNAs

Dmytro Ustianenko et al. RNA. 2013 Dec.

Abstract

The mechanisms of gene expression regulation by miRNAs have been extensively studied. However, the regulation of miRNA function and decay has long remained enigmatic. Only recently, 3' uridylation via LIN28A-TUT4/7 has been recognized as an essential component controlling the biogenesis of let-7 miRNAs in stem cells. Although uridylation has been generally implicated in miRNA degradation, the nuclease responsible has remained unknown. Here, we identify the Perlman syndrome-associated protein DIS3L2 as an oligo(U)-binding and processing exoribonuclease that specifically targets uridylated pre-let-7 in vivo. This study establishes DIS3L2 as the missing component of the LIN28-TUT4/7-DIS3L2 pathway required for the repression of let-7 in pluripotent cells.

Keywords: DIS3L2; RNA degradation; RNA uridylation; let-7 miRNA.

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Figures

FIGURE 1.

FIGURE 1.

Identification of Dis3l2 as an oligo(U)-binding nuclease in mouse embryonic stem cells (mESCs). (A) Schematic overview of the protocol. (B) Proteins identified in control and U30 RNA-bound nuclear extracts. (C) Proteins identified in control and U30 RNA-bound cytoplasmic extracts. Equal amounts of eluates from control RNA (Ctrl) and U30 RNA bait fractions were separated on 12% SDS-PAGE gels and silver stained. Proteins identified only in the U30 RNA sample are indicated on the right.

FIGURE 2.

FIGURE 2.

DIS3L2 is a cytoplasmic, oligo(U)-binding, Mg++-dependent exoribonuclease that does not associate with exosomes. (A) Schematic representation of the domain organization of DIS3L2 homologs from Saccharomyces cerevisiae and Homo sapiens. The PIN domain is shown in green, the CSD1 and CSD2 RNA binding domains are in orange, the RNB ribonuclease domain is in blue, and the S1 domain is in pink. (B) Immunofluorescence staining of HEK293T and HeLa cells transfected with either DIS3L2 C-terminally fused to an EGFP (DIS3L2-EGFP) or an empty EGFP-expressing plasmid (EGFP). DAPI was used to visualize nuclei. The scale bar corresponds to 10 μm. The Western blot on the right shows the relative abundance of endogenous and EGFP-tagged DIS3L2 in transfected HeLa cells as detected with a DIS3L2-specific antibody. (C) DIS3L2 does not interact with core exosome components. FLAG-tagged DIS3, DIS3L, and DIS3L2 were immunoprecipitated from stable cell lines inducibly overexpressing the individual proteins. The composition of the IP samples was analyzed by Western blot with the indicated antibodies. (D) DIS3L2 binds U30 RNA with nanomolar affinity. Electromobility shift assay with recombinant DIS3L2 and 5′ end-32P-labeled U30 RNA. The migration patterns of free RNA and protein-RNA complexes are indicated. (E) The catalytic activity of DIS3L2 requires divalent metal cations. A degradation assay using recombinant DIS3L2 with U30 RNA as a substrate was performed in the presence of different divalent metal ions as indicated. (EDTA) Reaction mixture containing 5 mM EDTA; (np) control reaction without the addition of any protein.

FIGURE 3.

FIGURE 3.

DIS3L2 targets 3′ end-uridylated precursors of let-7 miRNA. (A) Mutant DIS3L2 (D391N) specifically coprecipitates with extended forms of pre-let-7a miRNA. Northern blot analyses of RNAs coimmunoprecipitated with FLAG-tagged proteins as indicated on the upper side of the autoradiograph (IP-FLAG). The RNA input was total RNA isolated from whole cell lysates. The ethidium bromide (EtBr)-stained gels represent input loading controls. (Empty) Control RIP performed using cells that were not expressing any FLAG-tagged protein. The lower panel shows Western blot analysis of Myc-tagged LIN28A expression and the efficiency of FLAG tag-mediated immunoprecipitation of proteins used for RIP analyses. “Input” shows the whole cell lysates used for the IPs. The ectopic expression of Myc-tagged LIN28A was monitored with specific anti-Myc antibodies. DIS3L2 was detected with anti-FLAG antibodies. (B) Sequencing analysis of the 3′ termini of pre-let-7 RNAs coprecipitated with the D391N DIS3L2 mutant in HEK293T-Rex cells ectopically overexpressing LIN28A and pri-let-7a-1. The analysis suggests that the DIS3L2 mutant binds to uridylated pre-let-7a-1 and pre-let-7i because 31 out of 35 sequenced clones contained nontemplate oligo(U)-tails. The gray box schematically represents the body of pre-let-7 miRNAs. A graphical representation of the (U)-extension statistics is shown at the right. (C) Oligouridylation stimulates DIS3L2-mediated degradation of pre-let-7 miRNA. An in vitro degradation assay with FLAG-DIS3L2 purified from HEK293T-Rex cells and pre-let-7 RNA (represented by the schematic stem–loop) containing either no 3′ extension or 3′ oligo(U) and oligo(A) extensions of increasing lengths (indicated at the top) as substrates. D391N indicates a reaction with purified catalytically inactive DIS3L2. (D) DIS3L2 exhibits a preference for RNA substrates with oligo(U) extensions. A 5′ end-labeled pre-let-7-U8 RNA was incubated with purified DIS3L2 in the presence of a 10-fold excess of different unlabeled competitor RNAs (indicated at the top) for the time periods shown.

FIGURE 4.

FIGURE 4.

A model for the regulation of let-7 miRNA biogenesis via the LIN28A-TUT4/7-DIS3L2 pathway. The primary transcripts (pri-let-7) are processed by the microprocessor complex in the nucleus to pre-let-7, which is exported to the cytoplasm. In the cytoplasm, pre-let-7 is either processed by Dicer to mature let-7 miRNA, or it is oligouridylated in the presence of LIN28A. The oligo(U) tails are then recognized by the DIS3L2 exonuclease, which initiates the degradation of RNA in the 3′ to 5′ direction.

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