miRNA-mediated gene silencing by translational repression followed by mRNA deadenylation and decay

Abstract

microRNAs (miRNAs) regulate gene expression through translational repression and/or messenger RNA (mRNA) deadenylation and decay. Because translation, deadenylation, and decay are closely linked processes, it is important to establish their ordering and thus to define the molecular mechanism of silencing. We have investigated the kinetics of these events in miRNA-mediated gene silencing by using a Drosophila S2 cell-based controllable expression system and show that mRNAs with both natural and engineered 3' untranslated regions with miRNA target sites are first subject to translational inhibition, followed by effects on deadenylation and decay. We next used a natural translational elongation stall to show that miRNA-mediated silencing inhibits translation at an early step, potentially translation initiation.

Figure 1

Steady state evaluation of miRNA-mediated gene silencing using copper inducible in vivo reporter system. (A) Measured protein amounts (luminescence) from transfected non-targeted (NT), targeted (T) and control constructs 24 hours after induction. Additional expression of bantam miRNA, not Argonaute 1, results in increased repression for synthetic bantam targeted constructs (Supplementary Fig. 1). (B-E) Ratios of steady state protein amounts for synthetic and natural miRNA-targeted constructs 48 hours after induction. In each case, mean values ± standard deviation from three independent triplicate experiments are shown as a normalized ratio of protein amounts (NT/T).

Figure 2

Time resolved progression of miRNA-mediated gene silencing establishes that repression of protein synthesis precedes mRNA deadenylation and decay. (A-D) Normalized levels of protein amounts for both miRNA-targeted (T) and non-targeted (NT) constructs; normalized mRNA levels for reporter genes from oligo(dT)₂₅ resin pull down or from total RNA presented as ratios of poly(A) and total mRNA, respectively. Each data point represents the mean value ± standard deviation calculated from three independent experiments,

Figure 3

mRNA deadenylation is not required for miRNA-mediated translational repression. (A-D) Length of poly(A) tail was determined using G/I tailing PCR based amplification (Materials and Methods). Positions of over-amplified products with or without (C) poly(A) tail are indicated. GAPDH mRNA poly(A) tail length is shown as a control. M lane represents 100 bp markers. (E) Time resolved progression of miRNA-mediated gene silencing for histone H3 constructs. Normalized levels of protein and mRNA amounts for miRNA-targeted (T) and non-targeted (NT) constructs are shown. Each data point represents an average value from three independent experiments.

Figure 4

Translational elongation stalling assay indicates that miRNA-mediated gene silencing targets early steps in translation. (A-E) Time resolved progression of miRNA-mediated effects on the stability of various mRNA constructs containing the lysine-induced elongation stall. Normalized mRNA amounts for the miRNA-targeted (T) and non-targeted (NT) constructs are shown as the ratio of NT/T; note that values are less than one and decreasing. Each data point represents an average from three independent experiments.