Figure 5.

PAB-1 and PAB-2 are required for miRNA-mediated silencing independently of the poly(A) tail. (A) Design for the RNA used in the experiment. A capped transcript containing a Renilla luciferase ORF and six miR-35 binding sites in its 3′UTR, but lacking a poly(A) tail. (B) The reporter was incubated in an embryonic translation extract in the presence of 2′-O-Me inhibitor (miR-35) or non-specific inhibitor (miR-1). Translation activity was monitored through measurement of RL activity. Repression activity of the RL-6×-miR-35-p(A)₀ transcript was assayed in (C) GST- or (D) GST-PAIP2-treated extracts. Translation counts were monitored over time in the presence of miR-35 or miR-1 2′-O-Me inhibitors. (E) RL-6×-miR-35-p(A)0 was incubated in GST- or GST-PAIP2-treated extracts. RNA was extracted and analyzed by UREA-PAGE. *: significance in a one-sided Welch t-test.