Kinetic analysis reveals successive steps leading to miRNA-mediated silencing in mammalian cells

Abstract

MicroRNAs (miRNAs) regulate most cellular functions, acting by posttranscriptionally repressing numerous eukaryotic mRNAs. They lead to translational repression, deadenylation and degradation of their target mRNAs. Yet, the relative contributions of these effects are controversial and little is known about the sequence of events occurring during the miRNA-induced response. Using stable human cell lines expressing inducible reporters, we found that translational repression is the dominant effect of miRNAs on newly synthesized targets. This step is followed by mRNA deadenylation and decay, which is the dominant effect at steady state. Our findings have important implications for understanding the mechanism of silencing and reconcile seemingly contradictory data.

Figure 1

Construction and characterization of inducible cell lines. (A) Scheme of the genome-integrated reporters. RL miRNA-target reporters and a FL control reporter were cloned back-to-back under the control of the same tetracycline (tet)-responsive element. The indicated 3′UTRs in their wild-type (WT) form or containing mutated (MUT) miRNA-binding sites were fused to the RL coding sequence. Polyadenylation sites (SV40 or a 3′ long terminal repeat, LTR) are indicated. (B,C) Time course of RL and FL expression. CMV–RL–hmga2–WT and –MUT cell lines were seeded at the same density and induced for the indicated times. At each time point, the same number of cells was used for dual luciferase assay. Control FL reporters expressed in WT or MUT cell lines yielded similar readings (B), while the WT RL–hmga2 reporter was expressed at lower levels than the MUT RL–hmga2 reporter (C). Luciferase activity is expressed in arbitrary units. (D,E) Specificity of repression. Repression (ratio of normalized RL–hmga2–MUT over WT activities or mRNA levels) of the WT reporters was relieved on knockdown of the three TNRC6 proteins (D) or transfection of 2′-O-methyl oligonucleotides complementary to their targeting miRNA. As miR21 and miR590 share the same seed sequence, they were both targeted with specific anti-mirs (E). After 48 h transfection, cell lines were induced for 4 h before analysis. 0% indicates no effect on repression, 100%, no repression observed. Throughout this study, error bars represent 95% confidence intervals from at least three independent experiments done in duplicates. CMV, cauliflower mosaic virus; MMTV, mouse mammary tumour virus; 3′-UTR, 3′-untranslated region.

Figure 2

Translational repression precedes mRNA decay. (A) Detection of the earliest measureable protein and cytoplasmic reporter mRNA. CMV–RL–hmga2–WT and –MUT cell lines were induced for the indicated times. Cytoplasmic extracts were then used for luciferase and RNA analysis. The earliest detectable cytoplasmic RL–hmga2 mRNA and RL protein appeared at 60-min postinduction. RNA levels are normalized to glyceraldehyde-3-phosphate dehydrogenase. RL activity was adjusted to FL values. Background values at t=0 were subtracted. (B–D) Relative expression of wild-type RL reporters mRNA and protein over time. CMV–RL–hmga2–WT and –MUT (B), MMTV–RL–hmga2–WT and –MUT (C), and CMV–RL–reck–WT and –MUT (D) cell lines were induced for the times indicated. RL activity and cytoplasmic mRNA level (normalized to corresponding FL values) of the WT reporters are expressed as percentages of values for the matching MUT reporter, which are set to 100% at each time point (broken line). In (C), the asterisk at 1 h indicates that protein levels were too low for reliable measurements. CMV, cauliflower mosaic virus; FL, firefly luciferase; MMTV, mouse mammary tumour virus; MUT, mutated; RL, Renilla luciferase; 3′-UTR, 3′-untranslated region; WT, wild type.

Figure 3

Translational repression precedes mRNA deadenylation. (A) Expression of RL reporters after a 1-h transcription pulse. CMV–RL–hmga2–WT and –MUT cell lines were induced for 1 h. Transcription was then stopped with actinomycin D (ActD, arrow) and reporters analysed at the indicated time points for mRNA (left, normalized to GAPDH) and RL activity (right, adjusted for FL values, log₁₀ scale). (B) Estimation of poly(A) tail lengths by PCR analysis after polyG/I extension. Ao was obtained with reporter-specific primers amplifying a region upstream of the polyadenylation site. To measure poly(A) tail lengths, the reverse specific primer was replaced by a primer annealing to the poly(A)-polyG/I junction. Sizing of the PCR products was performed on a microfluidic chip. Poly(A) tail lengths estimated at different time points are indicated. (C) Relative levels of WT RL protein, mRNA, and fully polyadenylated mRNA after a 1-h transcription pulse. At each time point, RL activity and cytoplasmic mRNA level of the RL–hmga2–WT reporter are expressed as percentages of values for the matching MUT reporter. In addition, cytoplasmic RNA was fractionated according to poly(A) tail length (see text and supplementary Fig S4 online). The amount of mRNA present in the fraction containing reporters with the longest poly(A) tails (fraction E4 in supplementary Fig S4 online, termed ‘fully polyadenylated mRNA’) was calculated for both WT and MUT reporters. The percentage of fully polyadenylated WT reporter, relative to MUT reporter is indicated. The broken line at 100% indicates levels of the matching MUT (non-repressed) reporter (set to 100% at each time point). CMV, cauliflower mosaic virus; FL, firefly luciferase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MUT, mutated; RL, Renilla luciferase; WT, wild type.

Figure 4

Translational repression is independent of deadenylation. (A,B) Relative expression of RL–hmga2–WT protein over time. CMV–RL–hmga2–WT and –MUT cell lines were induced for the times indicated under control or deadenylation-impaired conditions. WT RL activity is presented as in Fig 2. (C) Expression of RL reporters after a 1 h transcription pulse. CMV–RL–hmga2–WT and –MUT cell lines were induced for 1 h. Transcription was then stopped with actinomycin D and reporters analysed at the indicated time points. (D) Poly(A) tail-length profiles of the RL reporters after a 1 h transcription pulse, chased for 2 h as in (C). (E) Analysis of knockdown efficiencies. Cell lines were transfected with siRNAs against CNOT1, 7, 8. Cytoplasmic extracts were analysed by western blot. The control proteins Ago2 and tubulin were not affected. (F) Sequence of events leading to miRNA-mediated repression as observed at steady state. The dashed line indicates that mRNA deadenylation may already start at 80 min postinduction. CMV, cauliflower mosaic virus; MUT, mutated; RL, Renilla luciferase; siRNAs, small interfering RNAs; WT, wild type.