See text for detailed discussion. miRNA-targeted mRNA decay is triggered by deadenylation followed by decapping. Pan2-Pan3 and Ccr4-Caf1 deadenylases form a super-complex that coordinates the two consecutive phases of deadenylation 35. For simplicity, cap-binding complex and its interaction with the 3' poly(A)-PABP complex are not depicted.

(a) Northern blots showing the effects of over-expressing λN-Ago2 (left) or λN-Ago2 (F2V2) (right) on deadenylation and decay of BBB+boxB. (b) Northern blots showing the effect of over-expressing λN-Ago2 (F2W2) (left) on deadenylation and decay of BBB+boxB and the effect of ectopically expressed Caf1 mutant (mut) on the decay of BBB+boxB tethered by λN-Ago2 (F2W2) (right). (c) Western blots showing the expression levels of the ectopically expressed proteins as indicated. α-tubulin served as a loading control. (d) Co-immunoprecipitation and Western blotting experiments showing that ectopically expressed Flag-TNRC6C and Flag-TNRC6B cannot effectively pull down ectopically expressed HA-Ago2(F2V2) or endogenous Caf1 deadenylase. Rabbit anti-Caf1 antibody was used at 1:4000 dilution. (e) Northern blots showing that tethering TNRC6C (upper right) to the otherwise stable BBB+boxB mRNA triggers highly processive deadenylation and rapid decay, which is impaired by over-expression of Caf1mut (lower left). Western blots (lower right) showing the expression levels of the ectopically expressed λN-TNRC6C and Caf1 mutant. α-tubulin served as a loading control. NIH3T3 B2A2 cells were transiently co-transfected with a Tet-promoter regulated plasmid encoding BBB+boxB and the plasmids encoding the proteins as indicated. Times correspond to hours after Tetracycline addition. The control mRNA (ctrl) and the preparation of Poly(A)⁻ RNA (A⁻) were as described in the legend to Figure 1.

(a) Diagram showing BBB and BBB+boxB mRNAs. The Ago protein N-terminally fused to a peptide derived from the N protein of bacteriophage-λ (λN-Ago) binds with high affinity to four boxB elements in the 3' UTR of BBB+boxB mRNA. (b) Northern blots showing that tethering Ago2 (middle) to the otherwise stable BBB+boxB mRNA triggers highly processive deadenylation and rapid decay, which is impaired by over-expression of Caf1mut (right). (c) Northern blots showing that expression of the control λN-lacZ did not enhance deadenylation or decay of BBB+boxB mRNA (left) and that deadenylation and decay of BBB mRNA lacking boxB was not accelerated in the presence of λN-Ago2 (right). (d) Northern blot showing that tethering Ago2 (H634P) mutant that lacks endonuclease activity enhances the deadenylation and decay of BBB+boxB mRNA. (e) Western blots showing the expression levels of the ectopically expressed λN-lacZ and λN-Ago2 or its mutant derivatives. α-tubulin served as a loading control. For panels b, c and d: times correspond to hr after tetracycline addition. The control mRNA (ctrl) and the preparation of Poly(A)⁻ RNA (A⁻) were as described in the legend to Figure 1.

(a) Diagram showing the physical map of β-globin mRNA carrying a sequence that perfectly matches let-7 miRNA (BBB+let-7per) and the 5’ decay intermediate (red arrow) produced through siRNA-mediated endonucleolytic cleavage within the perfect let-7 complementary site. (b) Northern blots showing that the rapid decay of BBB+let-7per mRNA can be triggered by endonucleolytic cleavage through siRMD or by deadenylation through miRMD (see the finer time points in the right). Solid red arrows point out the 5’ endonucleolytic products, open arrow points out the decay intermediates with a ~110 nt of poly(A) tail, and the solid arrowhead points out the decay intermediates with an oligo(A) tail. (c) Over-expression of mutant Caf1 (Caf1mut) impairs deadenylation and stabilizes a small portion of BBB+let-7per mRNA as a decay intermediate with a ~110 nt of poly(A) tail (pointed out by an open arrow). NIH3T3 B2A2 cells were transiently co-transfected with a Tet-promoter regulated plasmid encoding BBB+let-7per mRNA and the plasmid encoding Caf1mut. The α-globin/GAPDH mRNA was expressed constitutively and served as an internal standard for transfection efficiency and sample handling (ctrl). The times given at the top correspond to hr (left panels of b and c) or minutes (right panel of b) after tetracycline addition. Poly(A)⁻ RNA (A⁻) was prepared as described in the legend to Figure 1. The expression level of the ectopically expressed Caf1mut was analyzed by Western blotting (right panel of c). GAPDH served as a loading control.

(a) Diagram showing β-globin mRNA itself (BBB) or with 3 copies of either mutated let-7 (BBB+let-7mut) or wild-type let-7 (BBB+let-7wt) target sites and the biphasic deadenylation kinetics of BBB+let-7wt mRNA. (b) Northern blots showing deadenylation and decay of BBB, BBB+let-7wt, or BBB+let-7mut mRNAs. (c) and (d) Northern blots showing mRNA decay of the BBB+let-7wt mRNA in the absence (vector) or presence of ectopically expressed mutant (mut) Pan2, Caf1, or Dcp2 either alone (a) or in combination (b) as indicated below each panel. Open arrows indicate decay intermediates with a ~110 nt of poly(A) tail and solid arrowheads show decay intermediates with an oligo(A) tail. NIH3T3 B2A2 cells were transiently transfected with a Tet-promoter regulated plasmid encoding a reporter mRNA and the plasmids encoding Pan2mut, Caf1mut, and/or Dcp2mut as indicated. A plasmid encoding constitutively expressed α-globin/GAPDH mRNA was also co-transfected to provide an internal standard for transfection efficiency and sample handling (ctrl). The times given at the top correspond to hours (h) after Tetracycline addition. Poly(A)⁻ RNA (A⁻) was prepared in vitro by treating an RNA sample from an early time point with oligo(dT) and RNase H. (e) Western blots showing the expression levels of the ectopically expressed proteins. GAPDH served as a loading control.