Translational inhibition by deadenylation-independent mechanisms is central to microRNA-mediated silencing in zebrafish

Abstract

MicroRNA (miRNA) is a class of small noncoding RNA approximately 22 nt in length. Animal miRNA silences complementary mRNAs via translational inhibition, deadenylation, and mRNA degradation. However, the underlying molecular mechanisms remain unclear. A key question is whether these three outputs are independently induced by miRNA through distinct mechanisms or sequentially induced within a single molecular pathway. Here, we successfully dissected these intricate outputs of miRNA-mediated repression using zebrafish embryos as a model system. Our results indicate that translational inhibition and deadenylation are independent outputs mediated by distinct domains of TNRC6A, which is an effector protein in the miRNA pathway. Translational inhibition by TNRC6A is divided into two mechanisms: PAM2 motif-mediated interference of poly(A)-binding protein (PABP), and inhibition of 5' cap- and poly(A) tail-independent step(s) by a previously undescribed P-GL motif. Consistent with these observations, we show that, in zebrafish embryos, miRNA inhibits translation of the target mRNA in a deadenylation- and PABP-independent manner at early time points. These results indicate that miRNA exerts multiple posttranscriptional outputs via physically and functionally independent mechanisms and that direct translational inhibition is central to miRNA-mediated repression.

Fig. 1.

The Mid domain of TNRC6A is sufficient to induce translational repression and deadenylation. (A) Schematic structures of zebrafish TNRC6A and its deletion mutants. (B) Schematic representation of the λN tethering assay in zebrafish embryos. (C) Results of the tethering assay with TNRC6A fragments. The bar graph shows Rluc activity that was normalized to Fluc activity. The normalized Rluc activity with the HA-λN empty construct (HA-N) was set to one. The data show averages of three independent experiments. Error bars show SD. Asterisks indicate p < 0.01 compared to experiments with HA-N. (D) The qRT-PCR analysis of reporter mRNA stability. The normalized Rluc mRNA values [normalized to those of the HA-λN empty construct (HA-N)] were set to one. The data show averages of three independent experiments. Error bars show SD. (E) The poly(A) tail analysis of the Rluc-BoxB-pA reporter mRNA by RNaseH digestion and Northern blot. The lane +dT shows completely deadenylated fragments, which correspond to A0. (F) Western blotting detecting HA-tagged effecter proteins.

Fig. 2.

The Mid domain of TNRC6A represses translation via two motifs. (A) Schematic representation of the Mid domain of zebrafish TNRC6A. The two conserved motifs (PAM2 and P-GL) are shown. Sequence alignments of each motif comparing zebrafish TNRC6A, human TNRC6 proteins, and fly GW182 are shown. Conserved residues are marked with asterisks. Alanine substitutions introduced in the current study are shown on the bottom. (B) GST-pulldown assay detecting interaction between the GST-Mid domain and zebrafish PABP. A total of 10% of embryonic lysate was loaded as an input. PABP was detected using Western blotting (Upper). GST fusion proteins were visualized using CBB stain (Lower). (C) The results of the tethering assay with TNRC6A Mid domain mutants. The data were collected and are shown as described in Fig. 1C. (D) Western blot detecting HA-λN-tagged Mid domain proteins. The membrane was probed with anti-eIF5 antibody as a control. (E) The poly(A) tail analysis of the injected Rluc-BoxB-pA reporter mRNA using RNaseH digestion and northern blot at 0, 3 and 6 hours. The lane +dT shows a completely deadenylated fragment (A0).

Fig. 5.

miR-430 represses its target mRNA in the absence of PABP during zebrafish embryogenesis. (A) In situ hybridization detecting pabpc1a mRNA in a 24 hpf zebrafish embryo (purple). (B) Western blot detecting PABP protein in 24 hpf embryos injected with control MO or pabpc1a MO. The membrane was probed with anti-eIF5 antibody as a control. (C) The polysome profiles of control MO-injected (Upper) and pabpc1 MO-injected (Lower) zebrafish embryos at 24 hpf. (D) Analysis of miRNA-mediated target mRNA repression in the presence or absence of PABP. Bright field view (Upper panels), GFP fluorescence (Middle panels; green) and GFP mRNA (Bottom panels; purple) of 24 hpf zebrafish embryos expressing the GFP transgene with three copies of the imperfect target site for the ubiquitously expressed miRNA, miR-430 (miR-430 TS) or with mutated target sites (mut TS). Control MO (left columns) or pabpc1a MO (right columns) was injected as indicated. (E) Quantification of GFP expression levels in Fig. 5D. GFP intensity of the embryos with the mut TS and the control MO was set to one. Error bars show SD. Asterisks indicate p < 0.01 compared to the experiment with the mut TS and control MO. (F) The poly(A) tail analysis of the GFP mut TS mRNA with the control MO or pabpc1a MO. The lane +dT shows a completely deadenylated fragment (A0).

Fig. 3.

The Mid domain of TNRC6A represses translation via deadenylation-independent mechanisms. (A) The GST-pulldown assay detecting interactions between the GST-Mid domain and deadenylase components translated in rabbit reticulocyte lysate. Total of 1% of in vitro translation reaction was loaded as an input. Myc-tagged proteins were detected using Western blotting (Upper). GST fusion proteins were visualized using CBB stain (Lower). (B) Rluc reporter mRNA containing 5 copies of BoxB sites followed by the A98C10 tail. (C) The poly(A) tail analysis of the Rluc-BoxB reporter mRNAs at six hours, in the presence of control HA-λN peptide (HA-N) or the HA-λN tagged Mid domain (Mid). Left: The reporter mRNA with a normal poly(A) tail [Rluc-BoxB-poly(A)]. Right: The reporter mRNA with an A98C10 tail (Rluc-BoxB-A98C10). The lane +dT shows a completely deadenylated fragment (A0). (D) Tethering assay of the TNRC6A Mid domain with reporter mRNA containing the A98C10 tail in the presence of Myc-GFP. (E) Tethering assay of the TNRC6A Mid domain with reporter mRNA containing the A98C10 tail in the presence of Myc-Paip2. (F) Tethering assay of the TNRC6A Mid domain constructs with a reporter mRNA containing the 5′ ApppG cap without a poly(A) tail. Graphs in D, E, and F show the averages of three independent experiments. Error bars show SD.

Fig. 4.

miR-1 represses target mRNA in a deadenylation- and PABP-independent manner in zebrafish embryos. (A) Fluc reporter mRNA containing zebrafish pdlim1 3′UTR. Red boxes indicate the target site for miR-1. (B) Scheme of the miR-1 repression assay in zebrafish embryos. (C) The poly(A) tail analysis of the Fluc-pdlim1 3′UTR reporter mRNAs at six hours in the absence (−) or presence (+) of the miR-1 duplex. The left panel shows the reporter mRNA with a normal poly(A) tail [Fluc-pdlim1-poly(A)]. The right panel shows the reporter mRNA with the A98C10 tail (Fluc-pdlim1-A98C10). (D and E) The results of the miR-1 repression assay with reporter mRNA containing a normal poly(A) tail in the presence of control Myc-GFP. (F and G) Results of the miR-1 repression assay with reporter mRNA containing the A98C10 tail in the presence of control Myc-GFP. (H and I) Results of the miR-1 repression assay with reporter mRNA containing the A98C10 tail in the presence of Myc-Paip2. D, F, and H show normalized Fluc activity. E, G, and I show normalized Fluc mRNA levels, which were measured using qRT-PCR. The values of the experiments using miR-124 were set to one at each time point. The data shows averages of three independent experiments. Error bars show SD.