A role for the Perlman syndrome exonuclease Dis3l2 in the Lin28-let-7 pathway - PubMed (original) (raw)

A role for the Perlman syndrome exonuclease Dis3l2 in the Lin28-let-7 pathway

Hao-Ming Chang et al. Nature. 2013.

Abstract

The pluripotency factor Lin28 blocks the expression of let-7 microRNAs in undifferentiated cells during development, and functions as an oncogene in a subset of cancers. Lin28 binds to let-7 precursor (pre-let-7) RNAs and recruits 3' terminal uridylyl transferases to selectively inhibit let-7 biogenesis. Uridylated pre-let-7 is refractory to processing by Dicer, and is rapidly degraded by an unknown RNase. Here we identify Dis3l2 as the 3'-5' exonuclease responsible for the decay of uridylated pre-let-7 in mouse embryonic stem cells. Biochemical reconstitution assays show that 3' oligouridylation stimulates Dis3l2 activity in vitro, and knockdown of Dis3l2 in mouse embryonic stem cells leads to the stabilization of pre-let-7. Our study establishes 3' oligouridylation as an RNA decay signal for Dis3l2, and identifies the first physiological RNA substrate of this new exonuclease, which is mutated in the Perlman syndrome of fetal overgrowth and causes a predisposition to Wilms' tumour development.

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Figures

Figure 1

Figure 1. Dis3l2 is associated with uridylated pre-let-7

(a) Affinity-purified proteins analyzed by Coomassie blue staining and mass spectrometry. (b) Diagrammatic representation of Dis3l2 protein, Accession number (NCBI): NP_001165628.1. Cold-shock domains, Ribonuclease II domain (RNB), and S1 RNA-binding domain are indicated. aa, amino acids. The mutated catalytic Aspartic Acid is indicated. (c) Western blotting analysis of samples in (a) with indicated antibodies. (d–f) co-IP and Western blot analyses.

Figure 2

Figure 2. Dis3l2 preferentially degrades uridylated pre-let-7

(a) Flag-Dis3l2 incubated with different radiolabeled pre-miRNAs. Where indicated, 100ng competitor RNA was added to reduce background activity. (b) Flag-tagged Dis3l2 or mutant Dis3l2 incubated with pre-let-7a-1+14U (c) RNA degradation assay with a titration of Flag-Dis3l2. (d) His-Dis3l2 examined by Coomassie blue staining and Western blot. (e) Flag-Dis3l2 and His-Dis3l2 analyzed by Western blot and activity. (f) RNA degradation assay with a titration of His-Dis3l2. (g) Representative time course assay. (h) Quantitation of three independent experiments as in (g) with the corresponding calculated RNA half-lives. p<0.01 [two-way analysis of variance (ANOVA) test]. Arrows indicate radiolabeled pre-miRNA while arrowheads indicate degradation products.

Figure 3

Figure 3. Molecular determinants of Disl32 activity

(a) Reconstitution assays reveal longer Flag-uridine tails are preferred substrates for Dis3l2. (b) RNA degradation assays with in vitro transcribed pre-let-7 RNAs with indicated 3′ U-tail. (c) Schematic representation and Western blot of different Dis3l2 truncations used for (d) RNA degradation assays and (e) EMSA.

Figure 4

Figure 4. Dis3l2 is required for degradation of uridylated pre-let-7 in embryonic stem cells

(a) Western blot analysis of siRNA knockdown. (b) quantitative RT-PCR (q.RT-PCR) analysis of Dis3l1 knockdown. Error bars ±S.D. (n=3). (c) mature miRNA levels measured by q.RT-PCR. Error bars ±S.D. (n=3) and (d) Northern blot. (e) Dicer processing assay with Arrows indicating pre-miRNA and arrowhead indicating Dicer products. (f)(g) 1 μg of total RNA from the samples in (a) was fractionated into >200 nt and <200 nt long RNA and relative levels of pre-let-7 (f) or pri-let-7 (g) RNA were quantitated by real-time PCR. Error bars ±S.D. (n=3). (h) Western blot analysis of Dis3l2 cells. (i) Northern blot analysis of pre-let-7g.

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