Lin28 recruits the TUTase Zcchc11 to inhibit let-7 maturation in mouse embryonic stem cells

Abstract

Lin28 and Lin28B, two developmentally regulated RNA-binding proteins and likely proto-oncogenes, selectively inhibit the maturation of let-7 family microRNAs (miRNAs) in embryonic stem cells and certain cancer cell lines. Moreover, let-7 precursors (pre-let-7) were previously found to be terminally uridylated in a Lin28-dependent fashion. Here we identify Zcchc11 (zinc finger, CCHC domain containing 11) as the 3' terminal uridylyl transferase (TUTase) responsible for Lin28-mediated pre-let-7 uridylation and subsequent blockade of let-7 processing in mouse embryonic stem cells. We demonstrate that Zcchc11 activity is UTP-dependent, selective for let-7 and recruited by Lin28. Furthermore, knockdown of either Zcchc11 or Lin28, or overexpression of a catalytically inactive TUTase, relieves the selective inhibition of let-7 processing and leads to the accumulation of mature let-7 miRNAs and repression of let-7 target reporter genes. Our results establish a role for Zcchc11-catalyzed pre-let-7 uridylation in the control of miRNA biogenesis.

Figure 1. Identification of Zcchc11 as a candidate pre-let-7 TUTase

(a) Diagrammatic representation of mouse Zcchc11 protein, Protein accession number (NCBI): AAI50792.1. Poly(A) Polymerase (PAP) domains are depicted in light gray, Zinc finger CCHC domains are in black, Nucleotidyltransferase (NTr) domain is in dark gray. aa, amino acids. (b) EMSA performed using radiolabeled pre-let-7g. Both recombinant Lin28 (5 μM) and Flag-Lin28 immunoprecipitated from ES cells were analyzed for ribonucleocomplex formation with pre-let-7g. (c) In vitro pre-let-7 uridylation assay. Synthetic pre-let-7a-1 was incubated with Flag-Lin28, HA-Zcchc11, r.Lin28, or a combination of Lin28 and Zcchc11 proteins. rPAP protein (8 units) was used as a positive control.

Figure 2. Zcchc11 TUTase activity is Lin28-dependent

(a) Uridylation assays performed with pre-let-7g in the presence of unlabelled UTP, ATP, CTP or GTP. (b) TUTase assay performed with pre-let-7a with a catalytically inactive Zcchc11 protein. A mutant Zcchc11 protein with two mutated aspartate residues (D1008A, D1010A in the NTr) was used in the uridylation assay. c, Uridylation assay with synthetic pre-miR-21 as a substrate in the uridylation assay as in Fig 1(c).

Figure 3. Inhibition of Zcchc11 selectively elevates mature let-7 levels in embryonic cells

(a) Real-time PCR analysis of mRNA and miRNA levels in response to the indicated siRNA transfection of mouse P19 cells. Data is plotted as −ΔΔCt. Relative fold change equals 2^(−ΔΔCt). Positive and negative −ΔΔCt values reflect upregulation and loss, respectively. (b) Knockdown of Zcchc11 in mouse ES cells. Depicted errors bars are ±s.d. (c) Mature microRNA levels upon HA-Zcchc11 and HA-mutant Zcchc11 overexpression in P19 cells. Presented p-values are based on comparisons between control siRNA versus siRNA set for a given gene knockdown. Depicted errors bars are ±s.d.

Figure 4. Zcchc11 inhibition exclusively affects let-7 biogenesis and leads to repression of let-7 target gene expression

(a) The TaqMan Rodent MicroRNA A Array v2.0 was used to examine global miRNA levels. Independent siRNA sets were used as indicated in the axis legends and data plotted as −ΔΔCt. Left and right panels depict the results for Lin28 and Zcchc11, respectively. For each panel, the upper right quadrant contains those miRNAs that are reproducibly upregulated by both siRNA knockdown sets. Depicted errors bars are ±s.e.m. (b) Luciferase reporter assays to evaluate the in vivo relevance of elevated mature let-7 levels. Renilla luciferase lacked (termed “Absent”), contained an imperfect, or had a perfect let-7 binding site in its 3′UTR. Depicted errors bars are ±s.d.